Immunoglobulin reduced in thrombogenic agents and preparation thereof

ABSTRACT

The invention relates to an immunoglobulin composition reduced in thrombogenic agents and to methods for its preparation. One method comprises subjecting an immunoglobulin containing solution to a negative cation exchanger chromatography at a pH in the range of higher than 3.8 to equal to or lower than 5.3. The solution can also be subjected to a negative anion exchanger chromatography at a pH in the range of 7 to 8.2.

FIELD OF THE INVENTION

The invention relates to an immunoglobulin preparation comprising lowlevels of thrombogenic agents.

BACKGROUND OF THE INVENTION

Intravenous immunoglobulin (IVIG) preparations are increasingly used forthe treatment of a variety of immunological deficiencies and autoimmunedisorders including dermatomyositis, idiopathic thrombocytopenicpurpura, Kawasaki disease, and Guillain-Barré syndrome. A small numberof thromboembolic adverse events have been associated with the use ofWIG preparations (Brannagan et al. Complications of intravenous immuneglobulin treatment in neurologic disease. Neurology 1996; 47:674-677;Rosenbaum J T. Myocardial infarction as a complication of immunoglobulintherapy. Arthritis Rheum 1997; 40:1732-1733; and Dalakas M C. High-doseintravenous immunoglobulin and serum viscosity: risk of precipitatingthromboembolic events. Neurology 1994; 44:223-226).

These events, which include deep venous thrombosis and myocardialinfarction, have primarily been observed in patients receiving high-doseIVIG, and they have been attributed to an increase in blood viscosity(Dalakas M C. 1994; and Reinhart W H, Berchtold P E. Effect of high-doseintravenous immunoglobulin therapy on blood rheology. Lancet 1992;339:662-664).

Components of the contact system of blood coagulation have previouslybeen identified in human immunoglobulin preparations (Alving et al.Contact-activated factors: contaminants of immunoglobulin preparationswith coagulant and vasoactive properties. J Lab Clin Med 1980;96:334-346). Commercial preparations of immune serum globulin were shownto contain widely varying levels of prekallikrein activator (PKA) andkallikrein activity. These activities were of interest because of theirpotential to produce bioactive peptides that can increase vascularpermeability. The presence of vasoactive fragments of these proteins wasthought to be related to occasional adverse reactions duringadministration of immunoglobulin preparations. These authors also foundfactor XI (FXI) in immunoglobulin preparations (Alving et al. 1980).

In Alving et al. (1980) twenty-five lots of commercial Imune SerumGlobulins (ISG) were analyzed for PKA and kallikrein, components of thecontact activation system, which could mediate such reactions throughthe generation of kinins in recipients. Kallikrein activity ranged fromundetectable levels to >60% of the total potential kallikrein activityin normal plasma. PKA ranged from 5% to 3950% of the activity in areference plasma protein fraction that had caused hypotension. All butfive lots increased vascular permeability in the guinea pig. The fivelots which caused no increase were also the lowest in PKA and kallikreinactivity. When the immunoglobulin preparation was subjected to gelchromatography to separate the enzymatic contaminants fromimmunoglobulin G, only the fractions containing PKA and/or kallikreinincreased vascular permeability. Several lots of IVIG shortened thenonactivated partial thromboplastin time of normal plasma from 236seconds to 38 to 55 seconds. During gel chromatography, coagulantactivity was eluted in a position corresponding to a molecular weight of150,000; which was inhibited by antibody to human factor XI. These dataindicate that factor XIa is responsible for the procoagulant activityobserved and that PKA and/or kallikrein are potential mediators ofvasoactive reactions to WIG preparation.

Immunoglobins have also been found to contaminate factor XIpreparations. Factor XI and immunoglobulins co-purify through successiveion-exchange columns and require the addition of a specific concanavalinA affinity chromatography column to remove traces of IgG contaminationfrom factor XI (Bonno B N, Griffin J H. Human blood coagulation factorXI: purification, properties, and mechanism of activation by activatedfactor XII. J Biol Chem 1977; 252:6432-6437).

Wolberg et al. (Coagulation Factor XI Is a Contaminant in IntravenousImmunoglobulin Preparations. Am J Hem 2000; 65:30-34) demonstrated thata procoagulant, identified as activated factor XI, is present in IgGpreparations. In this study, twenty-nine samples of intravenousimmunoglobulin (IVIG) from eight different manufacturers were assayedfor procoagulant activity. Twenty-six of these samples shortened theclotting time of factor XI-deficient plasma. Of these, fourteen sampleshad factor XI activities greater than 0.001 U/ml of normal pooledplasma. The remaining samples possessed less than 0.001 U/ml of normalplasma activity. The procoagulant activity in these samples could beinhibited by an anti-factor XI polyclonal antibody, suggesting that theprocoagulant activity was factor XI. The procoagulant activity increasedin two samples after storage at 4° C. for 4 weeks, likely as a result offactor XIa autoactivation. Additionally, activity in some IVIG sampleswas able to directly activate factor IX, indicating that activatedfactor XI was present in these samples.

During the last 3 centuries numerous methods for purification ofintravenous immunoglobulin has been developed to meet the growing demandfor WIG. The vast majority of the manufacturing process includes varioustechnologies for capturing the immunoglobulin on a dedicated resinusually an ion exchange resin (Jerry Siegel. The Product: AllIntravenous Immunoglobulins Are Not Equivalent. The Journal of HumanPharmacology and Drug Therapy. Volume 25, Issue 11 Part 2, November2005). Capturing of immunoglobulin on a resin is very expensive and timeconsuming since it requires a large amount of resin. In average, everyliter of ion exchange resin captures about 30-50 g immunoglobulin.Therefore, when using a conventional large column with a capacity ofabout 100 L resin, 3-5 Kg immunoglobulin can be captured. Capturing theagents present in the immunoglobulin solution by a small column has aneconomical benefit.

U.S. Pat. No. 5,252,217 discloses a human Factor XI concentrate preparedby applying a cryoprecipitated plasma supernatant to afiltration-adsorption step and a single step of chromatography on cationexchange resin. The concentrate obtained is perfectly suitable fortherapeutic use in replacement therapy in cases of Factor XI deficiency.The cation exchange resin is equilibrated with a buffer solution at a pHof 5.5 to 6.5, and preferably a pH of 6.

U.S. Pat. No. 4,272,521 discloses a method for the removal of bothprekallikrein activator (PKA) and kallikrein-activatable precursor toPKA (Factor XII) from an immune serum globulin (ISG) solution using anion exchange material at a pH of ≧7.2.

U.S. Pat. No. 5,164,487 discloses a method of manufacturing anintravenously tolerable immunoglobulin-G preparation that is free ofaggregates, vasoactive substances and proteolytic enzymes. The startingmaterial is treated with 0.4 to 1.5% by volume of octanoic acid and thenchromatographed, especially on an ion or cation exchanger or hydrophobicmatrix.

United States Patent Application 2010/0311952 discloses a method forpurifying an immunoglobulin, wherein the method comprises applying anaqueous, buffered solution comprising an immunoglobulin in monomeric andin aggregated form to a cation exchange material under conditionswhereby the immunoglobulin in monomeric form does not bind to the cationexchange material, and recovering the immunoglobulin in monomeric formfrom the solution after the contact with the cation exchange material.

PCT application WO2010/072381 discloses a method for purifying animmunoglobulin, wherein the method comprises applying an aqueous,buffered solution comprising an immunoglobulin in monomeric, inaggregated, and in fragmented form to an anion exchange chromatographymaterial under conditions whereby the immunoglobulin in monomeric formdoes not bind to the anion exchange material, and recovering theimmunoglobulin in monomeric form in the flow-through from the anionexchange chromatography material, whereby the buffered aqueous solutionhas a pH value of from 8.0 to 8.5. In one embodiment the anion exchangechromatography material is a membrane anion exchange chromatographymaterial.

Jose et al. (2010) discloses that pasteurization during Immune GlobulinIntravenous production inactivates some thrombogenic agents such asclotting enzymes. However, this method does not selectively inactivatethe clotting enzymes and thus may alter the activity of theimmunoglobulin solution.

Thus, there is a need for a method which selectively removesthrombogenic agent from an immunoglobulin solution without affecting theimmunoglobulins.

SUMMARY OF THE INVENTION

The present invention relates to a method for specifically removing PKA,kallikrein and/or FXI(a) from an immunoglobulin preparation. In oneembodiment, the immunoglobulin preparation is derived from a blood orblood fraction.

In one aspect, the invention provides a method for removing athrombogenic agent from an immunoglobulin containing solution, themethod comprising the steps of: providing the immunoglobulin containingsolution at a pH in the range of higher than 3.8 to equal to or lowerthan 5.3; providing a support comprising immobilized negatively chargedgroups; contacting the solution with the support; and collecting anunbound fraction I.

In one embodiment of the present invention, the solution has a pH in therange of higher than 3.8 to equal to or lower than 5.0.

In another embodiment of the present invention, the solution has a pH inthe range of equal to or higher than 4.0 to equal to or lower than 5.0.

In another further embodiment of the present invention, the solution hasa pH in the range of higher than 3.8 to equal to or lower than 4.7.

Yet in another embodiment of the invention, the solution has a pH ofabout 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 4.7.

Yet in another further embodiment of the invention, the solution has apH in the range of higher than 3.8 to equal to or lower than 4.3.

In another embodiment of the invention, the solution has a pH in therange of equal to or higher than 4.0 to equal to or lower than 4.3.

In another further embodiment of the invention, the solution has a pH inthe range of equal to or higher than 4.1 to equal to or lower than 4.3.

In one embodiment of the present invention, the unbound fraction I isfurther contacted with the support comprising the immobilized negativelycharged groups under the same pH; and an unbound fraction II iscollected.

In another embodiment of the present invention, the support is in theform of a chromatographic material or a chromatographic membrane.

In another embodiment of the present invention, the support material ormembrane is hydrophilic and selected from the group consisting ofagarose, sepharose, acrylic beads, cellulose, controlled pore glass,silica gels, and dextrans; hydrophobic and selected from the groupconsisting of resins; or organic synthetic polymer selected from thegroup consisting of materials or membranes based on polyacrylamides orpolystyrens.

In one embodiment of the present invention, the negatively chargedgroups are immobilized to the support via a linker present between thesupport and the negatively charged groups.

In one embodiment of the present invention, the linker is selected fromthe group consisting of a protein, amino acid and peptide.

In one embodiment of the present invention, the support is chemicallymodified.

In one embodiment of the present invention, the support is a weak or astrong cation exchanger.

In one embodiment of the present invention, the immobilized negativelycharged groups are selected from the group consisting of derivatives ofsulfonic and other sulfur containing acids, formic and other carboxylicacids, phosphoric and other phosphorous containing acids, nitrate andother nitrogen containing acids, and a combination thereof.

In one embodiment of the present invention, the immobilized negativelycharged groups are sulfur containing acids such as sulfopropyl.

In one embodiment of the present invention, the immobilized negativelycharged groups are carboxylic acids such as carboxymethyl.

In one embodiment of the present invention, the method further comprisesthe steps of: adjusting the unbound fraction I or the unbound fractionII to a pH in the range of 7 to 8.2; contacting the unbound fraction Ior the unbound fraction II with a support comprising immobilizedpositively charged groups at a pH in the range of 7 to 8.2; andcollecting an unbound fraction.

In one embodiment of the present invention, the method further comprisesthe steps of adjusting and contacting the solution, prior to contactingwith the support comprising immobilized negatively charged groups, witha support comprising immobilized positively charged groups at a pH inthe range of 7 to 8.2; and collecting an unbound fraction.

In one embodiment of the present invention, the immobilized positivelycharged groups are selected from the group consisting of ammonium, alkylammonium, dialkylammonium, trialkyl ammonium, quaternary ammonium, alkylgroups, H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, amino functional group, and acombination thereof.

In one embodiment of the present invention, the immobilized positivelycharged groups are quaternary ammonium. The quaternary ammonium can beDiethylaminoethyl (DEAE).

Still in another embodiment of the invention, the method comprisescontacting the solution with the support comprising the immobilizedpositively charged groups at a pH in the range of 7 to 8.2; collectingthe unbound fraction; adjusting the pH of the unbound fraction to a pHin the range of higher than 3.8 to equal to or lower than 5.3;contacting the unbound fraction with the support comprising theimmobilized negatively charged groups at a pH in the range of higherthan 3.8 to equal to or lower than 5.3; and collecting the unboundfraction I.

Still in another further embodiment of the invention, the adjusting andcontacting the unbound fraction with the support comprising theimmobilized negatively charged groups is carried out at a pH in therange of higher than 3.8 to equal to or lower than 5.0.

In one embodiment of the present invention, the adjusting and contactingthe unbound fraction with the support comprising the immobilizednegatively charged groups is carried out at a pH in the range of equalto or higher than 4.0 to equal to or lower than 5.0.

In one embodiment of the present invention, the adjusting and contactingthe unbound fraction with the support comprising the immobilizednegatively charged groups is carried out at a pH in the range of higherthan 3.8 to equal to or lower than 4.7.

In one embodiment of the present invention, the adjusting and contactingthe unbound fraction with the support comprising the immobilizednegatively charged groups is carried out at a pH of about 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, or 4.7

In one embodiment of the present invention, the adjusting and contactingthe unbound fraction with the support comprising the immobilizednegatively charged groups is carried out at a pH in the range of higherthan 3.8 to equal to or lower than 4.3.

In one embodiment of the present invention, the adjusting and contactingthe unbound fraction with the support comprising the immobilizednegatively charged groups is carried out at a pH in the range of equalto or higher than 4.0 to equal to or lower than 4.3.

In one embodiment of the present invention, the adjusting and contactingthe unbound fraction with the support comprising the immobilizednegatively charged groups is carried out at a pH in the range of equalto or higher than 4.1 to equal to or lower than 4.3.

In another embodiment of the present invention, contacting the solutionwith the support comprising the positively charged groups is carried outat a linear velocity in the range of 1 to 2 ml/min/cm², and theimmunoglobulin containing solution has a temperature in the range of 8to 37° C.

In one embodiment of the present invention, the method further comprisesthe steps of: contacting the solution prior to contacting with thesupport comprising immobilized negatively charged groups with achromatographic material comprising three-dimensional cross-linkedhydrophobic acrylic polymer; and collecting an unbound fraction III.

In one embodiment of the present invention, the method further comprisesthe steps of: contacting the unbound fraction, unbound fraction I orunbound fraction II with a chromatographic material comprisingthree-dimensional cross-linked hydrophobic acrylic polymer; andcollecting an unbound fraction III.

In one embodiment of the present invention, the support comprisingimmobilized positively charged groups is a weak or a strong anionexchanger.

Another aspect of the invention relates to a method for preparing animmunoglobulin composition comprising the steps of: subjecting animmunoglobulin containing solution to at least two steps of negativechromatography: an anion exchanger chromatography at a pH in the rangeof 7 to 8.2; followed by a cation exchanger chromatography at a pH inthe range of higher than 3.8 to equal to or lower than 5.3.

In one embodiment of the present invention, the cation exchangerchromatography is carried out twice. In one embodiment, the two cationexchanger chromatography are carried out in tandem.

In one embodiment of the present invention, the cation exchangerchromatography is carried out at a pH in the range of higher than 3.8 toequal to or lower than 5.0.

In another embodiment of the present invention, the cation exchangerchromatography is carried out at a pH in the range of equal to or higherthan 4.0 to equal to or lower than 5.0.

In another further embodiment of the present invention, the cationexchanger chromatography is carried out at a pH in the range of higherthan 3.8 to equal to or lower than 4.7.

Yet in another embodiment of the present invention, the cation exchangerchromatography is carried out at a pH of about 4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, or 4.7.

Yet in another further embodiment of the present invention, the cationexchanger chromatography is carried out at a pH in the range of higherthan 3.8 to equal to or lower than 4.3.

Still in another embodiment of the present invention, the cationexchanger chromatography is carried out at a pH in the range of equal toor higher than 4.0 to equal to or lower than 4.3.

Still in another further embodiment of the present invention, the cationexchanger chromatography is carried out at a pH in the range of equal toor higher than 4.1 to equal to or lower than 4.3.

In one embodiment of the present invention, the method further comprisesa negative chromatography using a chromatographic material comprisingthree-dimensional cross-linked hydrophobic acrylic polymer.

In one embodiment of the present invention, the cation exchanger is inthe form of a membrane.

In one embodiment of the present invention, the cation exchangercomprises a sulfonic functional group.

In another aspect, the invention relates to an immunoglobulincomposition derived from blood or blood fractions, comprising 4%-10%protein and obtainable according to the method of the invention.

The invention also provides a receptacle containing the immunoglobulincomposition according to the invention.

In another aspect, the invention provides a method for treating asubject suffering from an immunodeficiency, an inflammatory disease, anautoimmune disease, or an acute infection, comprising administering tothe subject an effective amount of an immunoglobulin compositionaccording to the invention.

DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

The invention relates to a method for removing thrombogenic agents froman immunoglobulin preparation. In one embodiment of the invention, theimmunoglobulin preparation is derived from blood or blood fractions. Inanother embodiment, the blood or blood fractions are from more than onedonor or from a plurality of donors.

It was found according to the present invention that FXI and/or itsactive form FXIa can be effectively removed from an immunoglobulincontaining solution by subjecting the immunoglobulin solution to acation exchanger chromatography at a pH level of lower than 6. Optimalresults were obtained in a pH range of higher than 3.8 to equal to orlower than 5.3. This pH range was found to effectively remove FXIa andat the same time substantially preserve the immunoglobulin (IgG) levels.

These findings are surprising, in view that the immunoglobulin (havingan isoelectric point of 6-8 U.S. Pat. Nos. 6,592,905 and 4,296,027) hasa positive net electrical charge at a pH level of lower than 6 and assuch was expected to bind to the cation exchanger as well, therebyresulting in low immunoglobulin levels remaining in the solution (U.S.Pat. No. 6,592,905).

Without being bound by the mechanism, it appears that at a pH of lowerthan 6 FXIa and immunoglobulin compete for the negatively charged groupson the cation exchanger and binding of FXIa is more favorable.

Also, it was surprisingly found according to the present invention thata higher efficiency of FXIa removal from the immunoglobulin containingsolution can be achieved by subjecting the immunoglobulin solution tothe cation exchanger at a pH range of 4.0 to 4.3. These findings arealso surprising due to the fact that FXIa has a positive net charge in awide pH range and up to about pH 9 (its isoelectric point is 8.9-9.1;Haematologic Technologies Inc. Research Reagent Catalog). Surprisingly,these results were obtained either with a weak cation exchanger or witha strong cation exchanger.

It was also found according to the present invention that kallikrein(having an isoelectric point of 8.6-9.5) can be effectively removed froman immunoglobulin containing solution by subjecting the solution to acation exchanger chromatography at a pH level of 4.1-4.3 whilesubstantially preserving the IgG levels. These findings are surprisingin light of that the immunoglobulin has a positive net electrical chargeat this pH range and thus would have been expected to bind to the cationexchanger as well resulting in low immunoglobulin levels remaining inthe solution.

The results show maximal removal of a thrombogenic agent from an IgGcontaining solution, by subjecting the solution to a cation exchangerunder a very narrow pH range, while maintaining an unaltered level ofimmunoglobulin in the solution.

These findings paved the way to the development of a method for removinga thrombogenic agent from an immunoglobulin containing solution whilepreserving the majority of the immunoglobulin within the solution and/orwithout altering the IgG subclass distribution (e.g. IgG1—about 65%,IgG2—about 30%, IgG3—about 6%, and IgG4—about 1%). By “preserving themajority of the immunoglobulin within the solution” it is meant that themethod allows a recovery of equal or more than 75% of the immunoglobulinas compared to the initial immunoglobulin content before contacting thesolution with the support according to the invention. In one embodiment,the method allows a recovery of 80%, 85%, 90%, 95% and 99% of theimmunoglobulin as compared to the initial immunoglobulin content beforecontacting the solution with the support according to the invention.

The invention provides a method for removing a thrombogenic agent froman immunoglobulin containing solution, the method comprising the stepsof: providing the immunoglobulin solution at a pH in the range of higherthan 3.8 to equal to or lower than 5.3; providing a support comprisingimmobilized negatively charged groups; contacting the solution with thesupport; and collecting an unbound fraction. In one embodiment of theinvention, the solution has a pH in the range of higher than 3.8 toequal to or lower than 5.0. In another embodiment of the invention, therange is equal to or higher than 4.0 to equal to or lower than 5.0. In afurther embodiment, the range is higher than 3.8 to equal to or lowerthan 4.7, such a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or 4.7.Yet, in a further embodiment, the range is higher than 3.8 to equal toor lower than 4.3. Yet, in a further embodiment, the range is equal toor higher than 4.0 to equal to or lower than 5.3. In another furtherembodiment, the range is very narrow, of equal to or higher than 4.0 toequal to or lower than 4.3 or about 4.1 to about 4.3 and specificallyallows removal of FXIa and Kallikrein.

The term “thrombogenic agent” refers to an agent that has the potentialto induce fibrin clot formation. The term “thrombogenic agent” is usedherein interchangeably with the terms hemostatic, thrombotic andpro-coagulant agent. Thrombogenic agent may be, for example, kallikrein,FXI, FXIa, Factor XII, thrombin and PKA. The term “thrombogenic agent”includes thrombogenic induced agents and “thrombosis-generating agents”such as agents that activate thrombogenic factors in the coagulationcascade.

The term “support” as used herein includes a carrier, or any matrix usedto attach, immobilize, carry, or stabilize the negatively chargedgroups. Supports are well known in the art as described in Hermanson etal Immobilized Affinity Ligand Techniques (Academic Press Inc. 1992).The support for carrying out the method of the invention can be made ofany material which is capable of binding a molecule comprisingnegatively charged groups. Solid supports include, but are not limitedto, matrices, columns, coverslips, chromatographic materials, filters,microscope slides, test tubes, vials, bottles, ELISA supports, glass orplastic surfaces, chromatographic membranes, sheets, particles, beads,including magnetic beads, gels, powders, fibers, and the like.

In one embodiment of the invention the support is in the form of achromatographic material. In another embodiment of the invention, thesupport is in the form of a chromatographic membrane. The support can becomposed of a hydrophilic material such as agarose, sepharose, acrylicbeads, cellulose, controlled pore glass, silica gels, dextranes;hydrophobic material such as resins; or an organic artificial/syntheticpolymer such as materials based on polyacrylamides or polystyrens.Typical materials/polymers are commercially available under the tradenames Sephacryl® (Pharmacia, Sweden), Ultragel® (Biosepara, France)TSK-Gel Toyopearl® (Toso Corp., Japan), HEMA (Alltech Ass. (Deer-field,Ill., USA), Eupergit® (Rohm Pharma, Darmstadt, Germany). Also materialsbased on azlactones (3M, St. Paul, Minn., USA). Particularly preferredis Agarose® or Sepharose®. These materials are commercially available,for example, from Sigma, St. Louis. The chromatographic material can besuspended in an appropriate medium and the resulting slurry can be usede.g. in a chromatographic column. However, the method of the inventioncan also be carried out batch-wise e.g. by using a test tube or a batchreactor. In one embodiment of the invention the support is a FRACTOGEL®EMD, a TOYOPEARL®, or a TSK-GEL® polymer matrix. Column chromatographyis known in the art (Practical Protein Chromatography edited by Kenneyand Fowell Volume 11 Humana Press, 1992) and generally refers to atechnique in which a solution (the mobile phase) is allowed to traveldown a column and an individual component is being adsorbed by thestationary phase e.g. by the chromatographic material. The term“negatively charged groups” refers to a molecule comprising chemicalgroups which carry a negative charge such as derivatives of sulfonic andother sulfur containing acids (e.g. SO₄ ²⁻ and SO₃ ⁻), formic and othercarboxylic acids (e.g. COO⁻), as well as phosphoric and otherphosphorous containing acids (e.g. PO₄ ³⁻), nitrate and other nitrogencontaining acids (e.g. NO²) and combination thereof. In one embodimentof the invention, the negatively charged groups are sulfur containingacids. In another embodiment of the invention, the negatively chargedgroups are sulfopropyl (SP). In another further embodiment of theinvention, the negatively charged groups are carboxylic acids. Inanother embodiment of the invention, the negatively charged groups arecarboxymethyl (CM).

The support can have a hydrophobic or a hydrophilic surface that binds apart of the negatively charged groups by hydrophobic/hydrophiliccovalent interaction. The hydrophobic/hydrophilic surface of the supportmay also be a polymer such as plastic or any other polymer whereinhydrophobic/hydrophilic groups have been linked to such as polyethylene,polystyrene or polyvinyl. Alternatively, the negatively charged groupscan be covalently bound to the support via a linker bridging between thesupport and the negatively charged groups. The term “linker” as usedabove refers to a spacer arm or a leash having a molecular weight fromtens to million Daltons that is used as an intermediary connectorbetween the support and the negatively charged groups. E.g. the linkercan be a protein, a peptide and/or an amino acid. In case the supportbinds directly the molecule comprising the negatively charged groups(without a linker), a reactive group within the molecule comprising thenegatively charged groups, such as a hydroxyl group, an ester or anamino group or carboxy group may be used to join to a reactive grouppresent on the support in order to create the covalent bond. The supportmay also have a charged surface or can be modified to carry a chargedgroup that interacts with the negatively charged groups. The support mayhave other reactive groups that can be chemically activated so as toattach the negatively charged groups, for example, cyanogen bromideactivated matrices, epoxy activated matrices and the like. The supportmay also comprise an inorganic carrier such as silicon oxide material,e.g. silica gel, to which the charged groups can be covalently linked.In another embodiment, the charged groups are attached to a membranesurface or are incorporated into the membrane. The attachment of thecharged groups to the membrane can be carried out in the same manner asdescribed above.

The cation exchanger used according to the invention can be in the formof a membrane or in the form of a chromatographic material as definedabove.

The support comprising the immobilized negatively charged groups isgenerally referred to as a cation-exchanger. “Cation exchangers” arenamed for their ability to attract or bind cations or positively chargedparticles. Typically, the support system is negatively charged and amolecule will bind under a pH which renders the net charge of themolecule positive. Examples of commercially available cation-exchangersin the form of a membrane are Mustang® S membrane and Mustang® S capsulewhich comprise a sulfonic functional group.

In the case of using a membrane as the support (e.g. Mustang® Smembrane, Mustang® S capsule), the immunoglobulin solution may becontacted with the membrane at a fluid flow rate in the range of 2 to4.3 Membrane Volume (MV)/min. The term “fluid flow rate” normally refersto the flow of the solution through the support.

The term “isoelectric point” refers to the pH wherein a molecule carriesno net charge. Below the isoelectric point, the molecule carries a netpositive charge, above it the molecule carries a net negative charge.

The support comprising the negatively charged groups can be a weak (e.g.carboxymethyl (CM)-column) or a strong cation exchanger (e.g. Mustang® Smembrane). A weak cation exchanger generally refers to an exchangerwhich is comprised of a weak acid that gradually loses its charge as thepH decreases, while a strong cation exchanger generally refers to anexchanger which is comprised of a strong acid that is able to sustainits charge over a wide pH range.

The term “contacting” refers to any type of a combining action whichbrings the solution or fraction into sufficiently close contact with thesupport and more particularly with the charged groups of the support ina manner that a binding interaction will occur between the chargedgroups and any binding partner, e.g. a thrombogenic agent, present inthe solution/fraction. The solution/fraction can be incubated with thesupport for a sufficient period of time which allows binding between thecharged groups and the thrombogenic agent. The solution/fraction can bein a temperature in the range of 7° C. to 37° C. while contacting thesupport.

It was found according to the present invention that carrying out asecond cation exchanger chromatography step resulted in an increasedremoval of FXIa from the immunoglobulin containing solution as comparedto carrying out a single cation exchanger chromatography step. Thissecond cation exchanger chromatography step was carried out whilesubstantially preserving the IgG levels. Thus, the solution can becontacted with the support several times. Alternatively, when thesupport is a filter, more than one filter can be combined into a singlefunctional unit. In one embodiment of the invention, the firstfiltration step results in an unbound fraction I which is contacted withthe support comprising the immobilized negatively charged groups underthe same pH range specified above, and a second unbound fraction II iscollected.

The support can be equilibrated prior to contacting thesolution/fraction with a buffer e.g. by washing the support with thebuffer of the immunoglobulin containing solution.

The term “equilibrate” generally refers to allowing the column orsupport to reach a specific buffer condition such as a specific pH leveland/or ionic strength.

The term “un-bound fraction” typically refers to the flow throughmaterial obtained or collected following contacting the immunoglobulincontaining solution with a support comprising charged groups or to theflow through material that is obtained or collected following contactingan immunoglobulin containing solution/fraction with a chromatographysupport.

In one embodiment, the charged groups are negatively charged. In anotherembodiment, the flow through material obtained is referred to as“un-bound fraction I”. In another embodiment, “un-bound fraction I” issubjected to the support comprising the negatively charged groups, andthe flow through material is referred to as “un-bound fraction II”.

A chromatographic column can be prepared by packing a drychromatographic material or a pre-swollen material into a column. Drychromatographic material can be pre-treated by stirring the material in2 volumes of 0.5 N HCl (for obtaining a support comprising negativelycharged groups) or in 0.5 N NaOH (for obtaining a support comprisingpositively charged groups). The material can then be allowed to settlee.g. for about 30 minutes. The supernatant can be decanted and thematerial can be washed with H₂O until a pH value of 4 or 8,respectively, is reached. The material can then be suspended in 2volumes of 0.5 N NaOH (for a support comprising negatively chargedgroups) or in 0.5 N HCl (for a support comprising positively chargedgroups), and the supernatant can be decanted e.g. after 30 min.

The second suspension step can be repeated, and the column can then bewashed with H₂O until the filtrate is neutral.

A pre-swollen material (and the dry material treated as described in theabove paragraph) can be pre-treated by stirring the material for e.g. 5min with the buffer used for loading the immunoglobulin containingsolution in a ratio of 6 ml buffer/gram pre-swollen material (or 30 mlbuffer/gram dry material). The pH can then be adjusted to according tothe invention, and the material is allowed to settle e.g. for about 30min, and the supernatant is decanted. The slurry is then re-dispersed inequilibration buffer (ratio as above). For column packing, the materialis then allowed to settle.

Alternatively, a commercially available pre-packed ready-to-use-columncan be used without any pre-treatment.

Afterwards, the column (prepared from the dry material, pre-swollenmaterial, or the pre-packed ready-to-use-column) is packed, andequilibrated to the desired pH conditions (e.g. by using equilibrationbuffer).

After column equilibration, the immunoglobulin solution having thedesired pH is loaded and the column can be washed with about five bedvolumes equilibration buffer to wash out all unbound material. Thecolumn can be cleaned for a second use.

Preparation and pre-treatment of the chromatographic material, columnpacking, column equilibration, loading of the immunoglobulin containingsolution, collection of un-bound material, and column cleaning for asecond use are well known in the art see, for example, Protein liquidchromatography, Edited by Michael Kastner, Elsevier science B.V. 2000,pages 45-49.

The immunoglobulin containing solution can be subjected to a filtrationstep prior to chromatography in order to reduce aggregates in thesolution. The filtration can be carried out e.g. through a 1.2 μm depthfilter or 0.2 μm filter. In one embodiment of the invention, prior tocarrying out the anion exchange chromatography, the solution issubjected to 0.2 μm positively charged depth filter. Advantageously, thepositively charged depth filter is used to remove negatively chargematerials such as phospholipids, lipids and the like. An anion exchangerchromatography can be carried out using 7.5 L of resin e.g.diethylaminoethyl (DEAE) resin packed in a column e.g. having a diameterof 35 cm and a bed height of 15 cm. The packed column can beequilibrated with at least 48 column volume of Purified Water (PuW) orWater for Injectioion (WFI) at a fluid flow rate of 100-120 L/hour e.g.at a fluid flow rate 110 L/hour. After equilibration, 18 column volumeof immunoglobulin containing solution can be loaded into the column.Loading of the immunoglobulin containing solution can be carried out ata fluid flow rate of 50-70 L/hour e.g. at a fluid flow rate of 60L/hour. The solution can be at a temperature of 2-10° C. e.g. at 8-10°C. The chromatography can be carried out at room temperature (22±2° C.).

A cation exchanger chromatography can be carried out using a cationexchange filter membrane having a membrane volume of 1560 ml. Variouscombinations of membrane sizes can be used to obtain a final membranevolume of 1560 ml e.g. two filter membranes having a volume of 780 mlcan be connected in tandem to form a total membrane volume of 1560 ml.

The cation exchanger can be pre-conditioned with at least 38 membranevolume of 1 N NaOH followed by at least 64 membrane volume of 1 N NaClat a fluid flow rate of 1.6-2.3 L/min. The term “pre-conditioning”generally refers to washing the support or membrane prior to use inorder to remove un-wanted substances that may be present on the surfaceof the support or membrane. Afterwards, the filter membrane can beequilibrated with at least 64 membrane volume of 20 mM Sodium Acetate(adjusted to the desired pH) at a fluid flow rate of 1.6-2.3 L/min untilthe desired pH is achieved. After equilibration, at least 50-140membrane volume of immunoglobulin containing solution can be filteredthrough the filter membrane. The chromatography can be carried out atroom temperature (22±2° C.) at a fluid flow rate of 1.6-2.3 L/min. Thetemperature of the loaded solution can be 6-10° C.

The protein concentration in the immunoglobulin containing solution canbe in the range of 40 to 75 mg/ml during loading to the cation or anionexchangers e.g. a protein concentration of 45, 50, 55, 60, 65, 70, 75mg/ml.

In the case of column chromatography, an un-bound fraction can beobtained following washing of the loaded column with the same bufferused for equilibration and/or the buffer used for loading (oftentimesreferred as to “binding buffer”) of the immunoglobulin containingsolution onto the column.

It was also found according to the present invention that BenzamidineSepharose (which affinity binds serine proteases such as FXIa)affinity-chromatography was not suitable for removing FXIa from animmunoglobulin containing solution. However, it was surprisingly foundthat FXIa can be effectively removed from an immunoglobulin solution bysubjecting the immunoglobulin solution to heparin—affinitychromatography at a low pH level (e.g. a pH level of 5.3).

Without being bound by the mechanism it appears that at low pH levelsheparin—affinity chromatography acts as a cation-exchanger (havingacidic groups such as COOH and H₂SO₄) and thus can be advantageouslyused for effectively removing FXIa from an immunoglobulin containingsolution in accordance with the invention.

“Affinity chromatography” is generally based on a highly specificbiological interaction such as that between antigen and antibody, enzymeand substrate, or receptor and ligand.

In one embodiment of the invention, a FXIa removal of more than 75% e.g.80%, 85%, 90%, 95% and 99% from the immunoglobulin containing solutionor fraction as compared to the initial FXIa content (before contactingthe solution or the fraction with the support comprising the immobilizednegatively charged groups according to the invention) is consideredefficient.

In one embodiment of the invention, a kallikrein removal of more than75% e.g. 80%, 85%, 90%, 95%, 97% and 99% from the immunoglobulincontaining solution or fraction as compared to the initial kallikreincontent (before contacting the solution or the fraction with the supportcomprising the immobilized negatively charged groups according to theinvention) is considered efficient.

PKA has a zero net electrical charge at pH level in the range of 7-8.2(the isoelectric point of PKA is about 8; http://www.expasy.org) and assuch it is not expected to bind to positively charged groups.Nevertheless, it was found according to the present invention thatsubjecting an immunoglobulin solution with a pH level in the range of7-8.2 to an anion-exchanger resulted in effective PKA removal. Theresults also show that using a pH level range of 7-8.2 in ananion-exchanger chromatography also resulted in high IgG recoveries inthe un-bound fraction. A pH of 7-8.2 is a level wherein the IgG has zeroor negative net charge and thus, it was expected that some of it wouldbe lost due to binding to the anion exchanger. It was surprisingly foundthat subjecting an immunoglobulin containing solution to an anionexchanger at a pH level range of 7-8.2 resulted in maximal removal ofPKA and, practically, no loss of IgG.

Accordingly, in order to remove PKA and at the same time obtain a highimmunoglobulin recovery, the method according to the invention canfurther comprise a step of contacting the immunoglobulin containingsolution/fraction with a support comprising immobilized positivelycharged groups at a pH in the range of 7 to 8.2, and collecting theunbound fraction. Thus, the immunoglobulin containing solution issubjected to an anion exchanger at a pH in the range of 7 to 8.2.

In one embodiment, the method employs first the anion exchanger stepfollowed by the cation exchanger step of the invention. In anotherembodiment, the method employs first the cation exchanger step followedby the anion exchanger step.

In one embodiment, the pH of the immunoglobulin containing solution isadjusted with an acid or base solution to a pH in the range of 7 to 8.2and the solution is contacted with the positively charged groupspre-equilibrated with a buffer or PuW having the same pH as the solutionand the unbound fraction is collected. The pH of the collected fractioncan be adjusted with an acid solution to a pH in the range of higherthan 3.8 and equal to or lower than 5.3 and the fraction can becontacted with the negatively charged groups pre-equilibrated with abuffer having the same pH as the fraction and the unbound fraction iscollected.

In another embodiment, the pH of the immunoglobulin containing solutionis adjusted with an acid or base solution to a pH in the range of higherthan 3.8 and equal or lower than 5.3 and contacted with the negativelycharged groups pre-equilibrated with a buffer having the same pH as thesolution and the unbound fraction is collected. The pH of the collectedfraction can be adjusted with a base solution to a pH in the range of 7to 8.2 and contacted with the positively charged groups pre-equilibratedwith a buffer or PuW having the same pH as the fraction and the unboundfraction is collected.

In one embodiment of the invention, the anion and cation exchanger stepsare one immediately after the other. In another embodiment of theinvention, there are additional steps in between the anion and cationexchanger steps of the invention.

The steps of anion or cation exchanger can be carried out several timesto increase purification.

The term “positively charged groups” as used herein refers to a moleculecomprising chemical groups which carry a positive charge such asammonium, alkyl ammonium, dialkylammonium, trialkyl ammonium, quaternaryammonium [e.g. diethylaminoethyl (DEAE), a dimethylaminoethyl or atrimethylaminoethyl], alkyl groups, H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, aminofunctional group (e.g. NR₂H⁺), and combination thereof.

In one embodiment of the invention, the positively charged groups arequaternary ammonium (Q). In another embodiment of the invention, thepositively charged groups are Diethylaminoethyl (DEAE).

In one embodiment of the invention, the immunoglobulin containingsolution is first contacted with the support comprising the immobilizedpositively charged groups at a pH in the range of 7 to 8.2, the un-boundfraction is collected; then the un-bound fraction is contacted with thesupport comprising the immobilized negatively charged groups; and anun-bound fraction I is collected. Alternatively, the immunoglobulincontaining solution can be first contacted with the support comprisingthe immobilized negatively charged groups; an “un-bound fraction I” iscollected; and then un-bound fraction I is contacted with the supportcomprising the immobilized positively charged groups and an “un-boundfraction” is collected.

The support comprising the positively charged groups can be composed ofany material which is capable of binding a molecule comprising chemicalgroups which carry a positive charge as defined above.

The support comprising the immobilized positively charged groups isgenerally referred to as an anion-exchanger. “Anion exchangers” arenamed for their ability to attract or bind anions or negatively chargedparticles. Anion exchangers are well known in the art (Practical ProteinChromatography edited by Kenney and Fowell Volume 11 Humana Press,1992). In anion exchangers, the support system is positively charged anda molecule will bind if the buffer pH is higher than the protein'sunique isoelectric point.

The support comprising the immobilized positively charged groups can bea weak or a strong anion exchanger. A weak anion exchanger generallyrefers to an exchanger which is comprised of a weak acid that graduallyloses its charge as the pH decreases, while a strong anion exchangergenerally refers to an exchanger which is comprised of a strong acidthat is able to sustain its charge over a wide pH range.

The solution/fraction can be in a temperature in the range of 8 to 37°C. while contacting the support.

The immunoglobulin containing solution/fraction can be contacted withthe support several times and/or two or more filters combined into asingle functional unit can be used.

The support can be equilibrated prior to contacting the solution orfraction with the support e.g. by washing the support with the buffer ofthe immunoglobulin containing solution/fraction.

More specifically, following contacting the solution with the supportcomprising the immobilized positively charged groups, the un-boundfraction is collected (the flow through material obtained followingcontacting the immunoglobulin solution with the support). In the case ofcolumn chromatography, the un-bound fraction can be obtained followingwashing of the loaded column with the same buffer used for equilibrationand/or the buffer used for loading of the immunoglobulin containingsolution/fraction onto the column.

Alternative immobilizing possibilities between the support and thepositively charged groups are elaborated above for immobilizingpossibilities between the support and the negatively charged groups.

In the case of using a column as the support comprising the positivelycharged groups, the immunoglobulin containing solution can be contactedwith the column at a linear velocity between 1 and 2 ml/min/cm². Theterm “filtration linear velocity” refers to the velocity of a solutionthat flows through a column.

The invention also provides a method for removing a thrombogenic agentfrom an immunoglobulin containing solution comprising the steps of:providing the immunoglobulin containing solution at a pH in the range of7 to 8.2, providing a support comprising immobilized positively chargedgroups, and providing a support comprising immobilized negativelycharged groups; contacting the solution with the support comprising theimmobilized positively charged groups; collecting the un-bound fraction;adjusting the pH of the fraction to a pH in the range of higher than 3.8to equal to or lower than 5.3; contacting the un-bound fraction with thesupport comprising the immobilized negatively charged groups; andcollecting an unbound fraction I.

In one embodiment of the invention, detectable amounts ofthrombosis-generating agents (e.g. PKA, kallikrein and/or FXIa) areremoved from the immunoglobulin containing solution.

The term “detectable” refers, for example, to a level detected using amethod of analysis as described below in the Materials and Methodssection.

In one embodiment of the invention, a PKA removal of more than 80% e.g.83%, 85%, 90%, 95% and 99% from the immunoglobulin containing solutionor fraction as compared to the initial PKA content (before contactingthe solution or the fraction with the support comprising the immobilizedpositively charged groups according to the invention) is consideredefficient. The findings according to the invention also show that usingan SDR-column made of a three-dimensional cross-linked hydrophobicacrylic polymer resulted in removal of residual amounts of FXIa. AnSDR-column is a chromatographic technique in which the sample interacts,at a relatively high mobile phase salt concentration, with a hydrophobicstationary phase.

Accordingly, the immunoglobulin containing solution can be alsocontacted with a chromatographic material comprising three-dimensionalcross-linked hydrophobic acrylic polymer, and an unbound fraction (e.g.“unbound fraction III”) is then collected. Alternatively, the contactingwith the chromatographic material comprising the three-dimensionalcross-linked hydrophobic acrylic polymer can be carried out aftercontacting with the support comprising the immobilized negativelycharged groups or after contacting with the support comprising theimmobilized positively charged groups i.e. “un-bound fraction”,“un-bound fraction I”, or “un-bound fraction II” are contacted with thechromatographic material comprising the three-dimensional cross-linkedhydrophobic acrylic polymer, and an unbound fraction III is thencollected. An example of such three-dimensional cross-linked hydrophobicacrylic polymer is the HyperD resin supplied by Biosepra. HyperDchromatography involves a mixed-mode of adsorption of hydrophobicinteraction and molecular exclusion [Guerrier L et al. “Specific sorbentto remove solvent-detergent mixtures from virus-inactivated biologicalfluids”. J Chromatogr B Biomed Appl. 1995 Feb. 3; 664(1):119-125].

It was observed (using the Wessler animal Model as described in Wessleret al. Biologic assay of a thrombosis-inducing activity in human serum.J Appl Physiol. 1959; 14:943-946) that an immunoglobulin compositionsubjected to kallikrein, PKA and/or FXIa removal according to theinvention exhibited reduced thrombosis-inducing activity.

In one embodiment, the invention provides a method for purifying animmunoglobulin containing solution from thrombogenic agents comprising:subjecting the solution to at least two steps of negativeion-chromatography: an anion exchanger chromatography at a pH in therange of 7 to 8.2; followed by a cation exchanger chromatography at a pHin the range of higher than 3.8 to equal to or lower than 5.3. Themethod can further comprise the step of subjecting the solution tonegative hydrophobic chromatography using a chromatographic materialcomprising three-dimensional cross-linked hydrophobic acrylic polymer.

The invention also provides a method for preparing an immunoglobulincomposition comprising the steps of: subjecting an immunoglobulincontaining solution to at least two steps of negative ionchromatography: an anion exchanger chromatography at a pH in the rangeof 7 to 8.2; followed by a cation exchanger chromatography at a pH inthe range of higher than 3.8 to equal to or lower than 5.3.

The cation exchanger chromatography can be carried out at a pH range ofhigher than 3.8 to equal to or lower than 5.0, at a pH range of equal toor higher than 4.0 to equal to or lower than 5.0, at a pH range ofhigher than 3.8 to equal to or lower than 4.7, at a pH range of higherthan 3.8 to equal to or lower than 4.3, or at a pH range of equal to orhigher than 4.0 to equal to or lower than 4.3.

The methods of the invention can further comprise a negative hydrophobicchromatography using a chromatographic material comprisingthree-dimensional cross-linked hydrophobic acrylic polymer.

In certain embodiments, the flow through material from the cationexchanger according to the invention is named unbound fraction I; theflow through material from the anion exchanger according to theinvention is named unbound fraction; the flow through material from asecond step of cation exchanger according to the invention is namedunbound fraction II; and the flow through from a chromatographicmaterial comprising three-dimensional cross-linked hydrophobic acrylicpolymer according to the invention is named unbound fraction III. Eachof these fractions can be loaded in one or more of the chromatographicsteps and conditions according to the invention in different order toremove thrombogenic agents and to collect the immunoglobulin containingflow through fraction.

In one embodiment of the invention an immunoglobulin containing solutionis loaded into the three-dimensional cross-linked hydrophobic acrylicpolymer as described in U.S. Pat. No. 6,468,733. The resulting unboundfraction III is collected, the pH of the unbound fraction III isequilibrated to 7-8.2, and the fraction III is subjected to an anionexchanger under similar pH conditions. The unbound fraction iscollected, the pH of the unbound fraction is equilibrated according tothe invention before loading to a cation exchanger according to theinvention, and the unbound fraction I is collected. This last step canbe repeated.

The term “negative chromatography” refers to chosen conditions so thatonly a relatively small proportion (e.g. less than 25%) or none of apurified protein (i.e. immunoglobulin) binds to the chromatographysupport and it thus passes through the support in the chromatographicseparation. The predominant portion of the protein is thus present inthe flow through material.

The term “positive chromatography” refers to chosen conditions so thatthe majority of a purified protein (i.e. immunoglobulin) binds to thechromatography support and therefore a step of elution under nonisocratic conditions is required to recover the protein.

The results show that an efficient removal of thrombogenic agents wasobtained also in scale-up process of cation and anion exchangerchromatography.

The invention also provides an immunoglobulin composition comprising lowlevels of a thrombogenic agent. The immunoglobulin can be provided in aliquid or solid form e.g. as a lyophilized powder.

In one embodiment of the invention, the immunoglobulin containingsolution is first contacted with the support comprising the immobilizedpositively charged groups (anion exchanger); the resulting unboundfraction is collected and contacted with the support comprising theimmobilized negatively charged groups (cation exchanger) and theresulting unbound fraction I which comprises immunoglobulin compositionwith low thrombogenic agents is collected. In one embodiment of theinvention, the immunoglobulin containing solution is also contacted witha chromatographic material comprising three-dimensional cross-linkedhydrophobic acrylic polymer (hydrophobic chromatography-HIC).

An immunoglobulin composition with low levels of a thrombogenic agentrefers, for example, to a composition having less than 6 IU/ml PKA; to acomposition exhibiting a thrombin generation of less than about 100 nMwhen determining the formation of thrombin in the immunoglobulincomposition e.g. by carrying out a Thrombin Generation Assay asdescribed below; less than 0.8 ng/ml FXIa; and/or less than 200 ng/mlkallikrein (e.g. less than 126 ng/ml). PKA, kallikrein and FXIa levelscan be determined by carrying out an assay as described below in theMaterials and Methods section.

In another aspect the invention relates to an immunoglobulin compositionobtainable according to the method of the invention. The immunoglobulincomposition can be provided in a receptacle. The vial or pre-filledsyringe can contain different volumes of the composition, for example,the composition can have a volume of 0.5 ml, 2 ml, 10 ml, 30 ml, 50 ml,100 ml, 200 ml, 500 ml, 1 liter, 2 liter and 3 liter.

The term “receptacle” refers to any container designed for holding theimmunoglobulin composition.

The immunoglobulin composition can comprises a protein concentration inthe range of 2 to 20% w/v, or 5-10%. In one embodiment of the invention,the composition has a protein concentration in the range of 4.5-5.5%w/v. In another embodiment of the invention, the composition has aprotein concentration of about 5% w/v. In another embodiment, thecomposition has a protein concentration of about 10% w/v. In oneembodiment the protein concentration is about 50 mg/ml. The % ofimmunoglobulin out of total protein content can be above 90%. In oneembodiment of the invention, the percentage of immunoglobulin out oftotal protein content is 95%.

In another embodiment of the invention, the immunoglobulin compositioncomprises a low percentage of protein aggregates e.g. less than 3%protein aggregates.

The term “aggregates” refers to a chunk of material which containssolids such as protein aggregates. The aggregates can be measured byHPLC.

In one embodiment of the invention, the composition is provided in avolume of 50 ml and has a protein content of 2.5 g. In anotherembodiment of the invention, the composition is provided in a volume of100 ml and has a protein content of 5.0 g. In another embodiment of theinvention, the composition is provided in a volume of 200 ml and has aprotein content of 10.0 g.

Vials comprising the composition can be stored at a temperature of lowerthan 25° C., such as at a temperature of 0° C. or 2° C. to 8° C. andprotected from light until use.

The immunoglobulin composition can also comprise excipients. As usedherein the terms “excipient” refers to an inert substance which is addedto the pharmaceutical composition. The excipients can be added into thecomposition, for example, in order to ensure that the active ingredientretains its chemical stability and biological activity upon storage, toaid the manufacturing process and/or for aesthetic reasons e.g. color.Examples of excipients include, but are not limited to, various sugars,such as maltose or, D-sorbitol; glycine; polymeric excipients, such asPEG or serum proteins, such as albumin.

The immunoglobulin composition can comprise at least 95% Human NormalImmunoglobulin G as the active ingredient, 10% maltose and Water forInjection. The Immunoglobulin A (IgA) content can be equal to or less≦0.15 mg/ml.

Yet another object of the invention is accomplished by providing amethod for treating a subject suffering from an immunodeficiency e.g.primary and secondary immunodeficiency, an inflammatory disease, anautoimmune disease, or an acute infection, comprising administering tothe subject an effective amount of an immunoglobulin compositionaccording to the invention.

The term “subject” includes animals of mammalian origin, includinghumans. In one embodiment, the subject is a patient.

The term “an effective amount” refers to the dose required to prevent ortreat (relieve a symptom or all of the symptoms) a disease, disorder orcondition. The effective amount can be measured based on any change inthe course of the disease in response to the administration of thecomposition. The effective dose can be changed depending on the age andweight of the subject, the disease and its severity (e.g. early oradvanced stage) and other factors which can be recognized by the skilledin the art.

The immunoglobulin composition can be used for replacement therapy suchas in primary immunodeficiency (patients with primary defective antibodysynthesis such as agammaglobulinemia or hypogammaglobulinemia); ChronicLymphocytic Leukemia (CLL) with severe secondary hypogammaglobulinemiaand recurrent infections, in whom prophylactic antibiotics have failed;Myeloma in plateau phase with hypogammaglobulinemia and recurrentbacterial infections who have failed to respond to pneumococcalimmunization; Hypogammaglobulinemia I patients after allogeneichematopoietic stem cell transplantation (HSCT); Children with congenitalAIDS and recurrent infections; and Allogenic Bone MarrowTransplantation; and in Immunomodulation such as in Chronic inflammatorydemyelinating polyneuropathy (CIDP); Idiopathic Thrombocytopenic Purpura(ITP); Guillain Barré Syndrome; and Kawasaki Disease.

The dose and dosage regimen is dependent on the intended use. Inreplacement therapy, the dosage may need to be individualized for eachpatient, dependent on the pharmacokinetic and clinical response.

The immunoglobulin composition prepared according to the invention whichhas low levels of thrombogenic agents can be administered by routes thatlead to systemic absorption. Non limiting examples of administrationroutes include, but are not limited to, intravenous, subcutaneous,intraperitoneal, and intramuscular. Advantageously, patients may receivea high dose of the immunoglobulin solution prepared according to theinvention which has low levels of thrombogenic agents. Theadministration can be carried out in an initially higher dose e.g.0.4-0.8 g/kg followed by the same or lower doses at intervals. Thehigher doses can be intended to rapidly increase the patient'simmunoglobulin concentration to an efficacious target concentration.

The term “intravenous” refers to administration of the composition intothe vein of a subject. The administration can be intermittent or bycontinuous dripping. The term “intermittent” is synonymous with the term“intravenous bolus” or “intravenous push”.

The term “subcutaneous” refers to introduction of the composition byinjection under the skin of a subject. The injection can be carried outby creating a pocket such as by pinching or drawing the skin up and awayfrom underlying tissue. Optionally, the infusion may be carried out bysubcutaneous implantation of a drug delivery pump implanted under theskin of the subject. The pump can deliver a predetermined amount of theimmunoglobulin at a predetermined rate for a predetermined period oftime.

By “intramuscular” it is meant an introduction of the immunoglobulincomposition directly into a muscle. The injections can be given into anymuscle including, but not limited to, the deltoid, vastus lateralis,ventrogluteal and dorsogluteal muscles. The administration can becarried out at multiple locations. “Intraperitoneal injection” refers tothe injection of the immunoglobulin into the peritoneum.

The immunoglobulin composition of the invention can be prepared fromblood or blood fractions donated by healthy donors. The immunoglobulincan be prepared from pooled blood or blood fractions obtained from 1000donors and more. The immunoglobulin can be prepared from screened donorswith high titers of antibodies. An example of such a technique isdisclosed in WO-2007/017859 which content is incorporated herein byreference. The term “blood fraction” refers to a fraction of whole bloodwhich comprises immunoglobulins such as plasma or serum. Theimmunoglobulin composition can be obtained by re-suspending Paste II,from plasma fractionation e.g. according to Cohn fractionation and/orKistler-Nitschmann (KN) fractionation method.

Immunoglobulin compositions derived from blood components are typicallypurified from infective particles. The viral inactivation can be carriedout by filtration, nanofiltration, solvent/detergent treatment, heattreatment, such as, but not limited to, pasteurization, gamma or UVC(<280 nm) irradiation, or by any other method known in the art.

In one embodiment of the invention, the immunoglobulin composition ispurified by the solvent-detergent method using TnBP/Triton-X-100, and bynanofiltration at pH-4.

The term “infective particle” refers to a microscopic particle, such as,but not limited to, a microorganism or a prion, which can infect orpropagate in cells of a biological organism. The infective particles canbe viral particles.

The inactivation procedure of infective particles can be carried out byadding an inactivating molecule to the composition prior to and/orduring the purification procedure. The added molecules and theirproducts can be removed by gravitation, column chromatography or by anyother method known in the art. The removal of infective particles can becarried out by nanofiltration or by selective absorption methods such asaffinity, ion exchange or hydrophobic chromatography. A multi-step viralinactivation procedure can be carried out. For example, theimmunoglobulin containing solution can be subjected to solvent/detergenttreatment, heat treatment, selective chromatography and nanofiltration.

The term “viral inactivation” refers both to the situation whereinviruses are maintained in the solution but are rendered non-viable (forexample, by dissolving their lipid coat), and/or as to the situationwherein viruses are physically removed from the solution (for example,by size exclusion techniques).

“Solvent detergent (S/D) treatment” typically refers to a process thatinactivates envelope-coated viruses by destroying their lipid envelope.The treatment can be carried out by the addition of detergents (such asTriton X-45, Triton X-100 or Tween 80) and solvents [such astri(n-butyl) phosphate (TnBP), di- or trialkylphosphates]. Thesolvent-detergent combination used to deactivate lipid coated virusesmay be any solvent-detergent combination known in the art such as TnBPand Triton X-100; Tween 80 and Sodium cholate and others. Theconcentration of the solvent detergents can be those commonly used inthe art, for example, >0.1% TnBP and >0.1% Triton X-100. Typically, theconditions under which the solvent-detergent inactivates the virusesconsist of 10-100 mg/ml of solvent detergent at a pH level ranging from5-8, and a temperature ranging from 2-37° C. for 30 min. to 24 hours.However, other solvent detergent combinations and suitable conditionswill be apparent to any person versed in the art. The bulk of thesolvent-detergent used in the S/D treatment can be removed, for example,by using chromatography columns such as hydrophobic interactionchromatography column (HIC) e.g. C-18 silica packing material and SDR(Solvent-Detergent removal) HyperD; protein adsorption matrices such asion-exchange matrices; affinity matrices; and/or size-exclusionmatrices. The S/D removal can further comprise a step of oil extraction.

“Nanofiltration” typically refers to a process by which lipid-envelopedand non-enveloped viruses are excluded from the solution e.g. by usingspecial nanometer-scale filters such as Planova™ 20N, 35N and 75N;Viresolve/70™, Viresolve/180™. The filters can have a pore size of lessthan 70 nm, preferably between 15 and 50 nm. However, any membranehaving a pore size sufficient to reduce or eliminate viruses from thesolution can be employed in nanofiltration. Viruses removed bynanofiltration can be enveloped [e.g. HIV, hepatitis B virus, hepatitisC virus, West Nile Virus, cytomegalovirus (CMV), Epstein-Barr virus(EBV), herpes simplex virus], and non enveloped (e.g. hepatitis A virus,paravirus B19, Polio virus).

Examples of immunoglobulin purification techniques are disclosed in U.S.Pat. No. 6,468,733, EP patent No. 1,161,958 and International PCTPublication WO 99/18130 whose contents are incorporated by reference.For example, a method for the purification of immunoglobulins from asource solution such as Cohn Fraction II may comprise: (a) pre-treatinga cation exchange resin with an acidic solution having a pH of 4.0-4.5;(b) contacting the source solution with the cation exchange resin; and(c) eluting the immunoglobulins bound to the cation exchange resin.Prior to contact with the cation exchange resin, the source solution maybe treated with an organic solvent and detergent.

Another method for the purification of immunoglobulins may comprise: (a)treating the solution with a solvent-detergent combination, atconcentrations and under conditions which are sufficient to inactivatelipid-coated viruses; (b) removing the solvent-detergent combinationfrom the solution by passing the solution obtained in (a) on achromatographic packing composed of silica beads which pore volume isfilled with three-dimensional cross-linked hydrophobic acrylic polymer;and (c) passing the solution of step (b) through a filter having a poresize from about 15 nm to about 70 nm as described in U.S. Pat. No.6,468,733.

The immunoglobulin composition can be concentrated by ultra-filtrationprocess. The ultrafiltration can be followed by diafiltration toexchange the buffer. The concentration and dialysis by ultrafiltrationand diafiltration, respectively, can be performed in one step or as twoseparate steps. The diafiltration can be carried out against anysolution which is suitable for human administration. Non limitingexamples of such solutions include, but are not limited to, 0.3% NaCland from about 1.6 to about 2.6% glycine such as about 2.25%.

EXAMPLES Materials and Methods

Immunoglobulin Solution.

a) Paste II Resuspension.

Paste II (45 Kg), prepared from plasma by the Cohn fractionation method(Cohn, E. J. The history of plasma fractionation. In Advances inMilitary Medicine, Andrus et al. Eds. Little, Brown & Co, 1948), wastransferred to a crusher tank to which cold (4° C.) Water for Injection(WFI) (3 times the weight of paste II) were added to achieve a finalweight of 180 Kg. The re-suspended paste was first stirred for a periodof about 5 hours at 4-6° C., and then decanted at 4-6° C. withoutstirring for about 37 hours, to allow precipitation of major proteinaggregates. Following precipitation the supernatant was separated fromthe precipitate and was used for the following step.

b) CUNO Filtration

The supernatant obtained in the previous step was filtered through aparallel pair of CUNO 0.2 μm positively charged depth filters (Cuno ZetaPlus, Cuno incorporation Inc. CT USA), in order to remove aggregates.The filter pressure did not exceed 1.0 Bar during the filtration step.The fluid flow rate during the filtration was 60 L/h and the temperaturewas 7.2° C.

c) Anion-Exchange Chromatography—Diethylaminoethyl Cellulose(DEAE)-Column.

In the next step, the solution resulting from step b) was subjected toan anion exchanger (see Examples 7-11).

Unless otherwise indicated, the DEAE-column was equilibrated with 7column value (CV; i.e. 77 ml) of Pure Water (or WFI) at a fluid flowrate of 2.8 ml/min. After equilibration, the immunoglobulin solutionresulting from step b) was loaded into the column. The column pressuredid not exceed 1.0 Bar.

In the case of further processing, step c) unless otherwise indicated,was carried out using the following conditions (scale-up conditions):DEAE-column (Toyopearl DEAE-650M; TOSOHAAS) was used as the anionexchanger [7.5 L resin. The column's diameter was 35 cm; and a bedheight of 15 cm]. The column was washed with 360 L of WFI at a fluidflow rate of 110 L/hour. The loading was carried out as a continuationto the CUNO filtration at a fluid flow rate of 60 L/hour at 8-10° C.(material temperature). The loading volume of the immunoglobulinsolution was 18 column volumes, the pH of the subjected solution was7-7.6. The Chromatography was carried out at room temperature (22±2°C.).

d) pH Adjustment

The pH of the solution was adjusted to 4.59 by adding 0.5 M HCl undercontinuous agitation. The temperature was maintained at 8±2° C. In orderto remove aggregates, the solution was filtered through 0.45 μm-0.65 μmfilter (Sartobran) into a clean vessel.

e) Concentration to 90 g/L and Diafiltration

The resulting solution of d) was ultra filtered by using anultra-filtration cassette containing a membrane with an exclusion limitof 30,000 D (Filtron Maxisette; 30 KD). The cassette was prepared bywashing with 500 Kg WFI. During the ultrafiltration, the proteinsolution (200 Kg) was concentrated to 90 g/L (a final weight of 141 Kg).This step was followed by diafiltration against 705 Kg WFI at constantvolume in order to remove the residual ethanol and to decrease theosmolality to <30 mOsm/Kg. The protein concentration was then adjustedto 73 g/L with WFI to reach a final volume of 173 Kg. The temperaturewas maintained at 8±2° C. throughout this step.

Cation-exchange chromatography [by using Mustang® S membrane, Mustang® Scapsule, SP-column or CM-column (for the specific conditions seeExamples 2-4, Example 5, Example 1, and Example 1, respectively)] wascarried out following step c)—using SP-column or following step e)—usingMustang® S membrane or Mustang® S capsule.

When the cation-exchange chromatography was carried out following stepc)—about 50-60 mg/ml protein were subjected to the chromatography. Whenthe cation-exchange chromatography was carried out following stepe)—about 70 mg/ml protein were subjected to the chromatography.

In order to reduce aggregates, prior to subjecting the immunoglobulinsolution to the cation-exchanger the solution was filtered through afilter [1.2 μm depth filter obtained from Sartorius sartopure (prior toSP-column) or 0.2 μm CA filter obtained from Corning (prior to Mustang®S membrane or Mustang® capsule)].

For SP-column, the column was equilibrated prior to loading theimmunoglobulin solution with 20 ml of 20 mM Acetate buffer having acompatible pH level as the immunoglobulin solution to be loaded.

For Mustang® S membrane, the filter membrane was equilibrated prior toloading the immunoglobulin solution with 20 ml of 20 mM Acetate bufferhaving a compatible pH level as the immunoglobulin solution to beloaded.

For Mustang® S capsule, the capsule was pre-conditioned beforeequilibration according to the manufacturer's instructions. Unlessotherwise indicated, in the next step, the capsule was equilibrated with600 ml of 20 mM Acetate buffer having a pH level of 4.2.

Solvent/Detergent (S/D) Treatment.

The immunoglobulin solution was equilibrated to pH of 5.3 and subjectedto S/D treatment (to inactivate lipid-enveloped viruses) as follows: 1%Triton X-100 and 0.3% tri(n-butyl) phosphate (TnBP) (v/v) were mixedtogether and then added slowly into the solution while rapidly stirring(20 Hz) the solution. The solution was then incubated for about 4.5hours at 6.9° C., under constant, gentle stirring. At the end of theincubation period, the temperature was raised to 23° C. over a period of1-1.5 hours and under agitation (at a speed of 20 Hz). In the next step,the SD-treated solution was sequentially filtered through a 3μ depthfilter (Sartorius) followed by a 0.45-0.65 μm membrane filter(Sartorius) (in order to remove gross particulate debris prior to asubsequent step of S/D removal).

S/D Removal by SDR-Column.

Removal of the S/D was carried out by a dedicated column of HyperDsolvent-detergent removal chromatography resin (SDR-HyperD by Biosepra).Prior to loading the SD-treated immunoglobulin solution, the column wasprepared with 450 Kg of WFI (at a maximal pressure of 0.8 bar) and at aflow rate of 80 L/h. The column length was 54 cm, with a diameter of 28cm and the resin's volume was 30-32 L. The flow rate was 78 L/h, and 37Kg of WFI was used to wash the column after loading of the sample toachieve baseline. The total flow through volume collected was 176.7 Kg.

Measurements of Prekallikrein Activator Levels.

Prekallikrein Activator (PKA), the first zymogen in the intrinsiccoagulation cascade, activates Prekallikrein to Kallikrein. In thisassay, the Prekallikrein (which is added to the tested sample) isactivated to Kallikrein by PKA found in the tested sample. The formedKallikrein then cleaves a Chromogenic substrate (H-D-But-CHA-Arg-pNA) tocolored p-nitroaniline (pNA) at a constant rate (see the reactionbelow). The reaction can be measured spectrophotometrically at 405 nm.

The obtained color is proportional to the amount of PKA present in thetested sample.

All the required reagents are from the Prekallikrein Activator Assay Kit(Pathway Diagnostics; Product Code: PW30100). The assay was carried outat 37° C.

More specifically, the assay was carried out as follows:

i) Step A for blank preparations: 25 μl of each PKA standard dilutions(0, 3.125, 6.25, 12.5, and 18.75 IU/ml. The various PKA concentrationswere prepared from a PKA solution diluted with the buffer's kit);positive control [an FDA International Standard for PKA which contained10 IU/ml PKA]; or diluted test samples (1:2 dilution with the buffer'skit) were pipetted into a 96-well microplate.

ii) Steps for PKA standards, positive control and test samples: 25 μl ofeach PKA standard dilution or diluted test samples (the same dilutionsas prepared above) were pipetted into an eppendorf tube. Then, 50 μl ofHuman Prekallikrein solution was added into each eppendorf tube. Thetube was capped and mixed and 25 μl from each eppendorf tube wastransferred (in duplicates) into microplate wells.

iii) In the next step, the plate was incubated for 30 minutes at 37° C.

iv) Step B for blank preparations: After 30 minutes incubation, 50 μl ofa pre-heated (37° C.) Human Prekallikrein solution was added into allblanks (see preparations of step i), and the content of the well wasmixed using a pipette. Then, 25 μl of the content of each of these wellswas immediately transferred (in duplicates) into correspondingmicroplate wells. The microplate was incubated at 37° C. for additionalabout 15 min (a total of 45 min incubation period).

v) Steps for all preparations: 100 μl of pre-warmed (at 37° C.)Kallikrein substrate working solution (a solution comprising theChromogenic substrate) were added to all the wells (wells of steps iiand iv).

vi) The microplate was placed in an ELISA-reader (at 37° C.) and theoptical density (OD) was measured at 405 nm after 2 min (OD_(2 min)) andonce more after 12-17 minutes incubation (OD_(X min)).

The OD obtained for the blanks were subtracted from the OD obtained forthe correspondent test samples.

The PKA content was calculated using ΔOD_((x-2)) (following subtractionof the blank from each reading) as interpolated from a standardcalibration curve taking into account the relevant dilution factor. Theresults are presented in International Units/ml.

Thrombin Generation Assay—for FXI/FXIa Removal Estimation.

Thrombin Generation Assay (TGA) is a global haemostatic method measuringthe amount of thrombin generated and degraded over time. This assayestimates the capacity (or potential) of any given sample to generatethrombin when the coagulation cascade is triggered (either by anintrinsic or an extrinsic trigger). TG is monitored via the conversionof a fluorogenic thrombin substrate and calibration of the thrombingeneration of the sample against a defined thrombin activity standard.

The assay was carried out in transparent round bottom (U-bottom) 96-wellplates and Thermo Electron Fluorometer (“Fluoroskan FL”) equipped with a390/460 nanometer filter set and a dispenser.

In order to measure FXI/FXIa levels, FXI deficient plasma (obtained fromStago; Catalog Number 00723) was used and the coagulation cascade wastriggered by 1 pM Tissue Factor (leads to activation of the extrinsiccoagulation pathway) and 4 μM phospholipids (leads to activation of theintrinsic coagulation pathway) (Tissue Factor and phospholipids aremixed together and provided in one reagent-PPP-Reagent low; Stago;Catalog Number TS 31.00).

The measurement was carried out according to: “The Thrombogram Guide”(Thrombinoscope BV): outline of the method to measure thrombingeneration using the Calibrated Automated Thrombogram” with thefollowing modifications:

For the measurement, 20 μl of the tested sample (serving as a potentialsource of FXIa) was mixed with 60 μl FXI deficient plasma, and 20 μlPPP-Reagent low in the well.

Each individual sample tested requires a corresponding calibrator sample(which comprises a known amount of thrombin) and the tested sample. Forthe thrombin calibrator sample measurement, 20 μl of the tested sample,60 μl FXI deficient plasma, and 20 μl thrombin calibrator (Stago;Catalog Number TS 20.00) were mixed.

The thrombin generation reaction (at 37° C.) was initiated upon additionof 20 μl containing the fluorescent substrate and Ca²⁺ [the fluorescentsubstrate and Ca²⁺ are provided in Fluca-kit (Stago; Catalog Number TS50.00) and are mixed together according to the manufacturer'sinstructions]. The addition of the fluorescent substrate and the Ca²⁺ iscarried out automatically by the “Fluoroskan FL”.

The amount of generated Thrombin (in nM) was then calculated using theinstrument software Thrombinoscope BV (provided with the “FluoroskanFL”). The final thrombin amount in the tested sample was calculated byreducing the background value from both the tested sample's value andthe thrombin calibrator's value and extrapolating the thrombin amount inthe sample from the thrombin calibrator value. The background value isobtained with 60 μl FXI deficient plasma supplemented with 20 μl buffercontaining 20 mM Acetate buffer.

Measurements of Factor XIa (FXIa) Levels (Fluorogenic Method).

Factor XIa levels were measured by a fluorogenic method. In the method,FXIa cleaves a specific fluorogenic substrate in the presence ofcalcium. In the cleavage reaction the substrate (which is composed froma fluorescent reporter group 6-amino-1-naphthalene-sulfonamide (ANSN)attached to a tri-peptide sequence) is hydrolyzed between thetri-peptide sequence and the ANSN group. Once cleaved from the peptidemoiety, the ANSN group exhibits about a 1000-fold increase in relativefluorescence. This activity is directly related to the amount of FactorXIa in the sample. The developed fluorescence is measured by using anELISA reader with the following wavelengths: excitation at 350 nm andemission at 470 nm. More specifically, the measurement was carried outas follows:

First stage (1^(st) 96-well plate preparation)—Tested samples weretested either undiluted (in the case the sample is tested aftersubjecting the immunoglobulin solution to the cation exchanger ordiluted (1:10 in the case the sample is tested before subjecting theimmunoglobulin solution to the cation exchanger). The dilution wascarried out in buffer B [40 mM Hepes, 300 mM NaCl, 4 mM CaCl₂, 0.2%polyethylene glycol (PEG) 20 K solution]. 50 μl from the above mentionedsamples were added into a well of a 96 well-plate (Costar; CatalogNumber 3797) in quadruplicate.

A standard curve was prepared using FXIa (Hematological TechnologiesInc.; Catalog Number HCXIA—0160) diluted in buffer A [20 mM Hepes; 150mM NaCl solution; 0.1% (w/v) BSA] to the following concentrations: 140,100, 70, 50, and 25 ng/ml. 50 μl of each concentration was added intothe wells in quadruplicate. Positive control (an immunoglobulinpreparation which contained about 120 ng/ml of FXIa) and blank samples(Buffer A) were added to each plate in quadruplicate (50 μl of eachpositive and blank samples).

Second stage (2^(nd) 96-well plate preparation)—For the kinetic reactiona second 96-well plate was prepared (Costar; Catalog Number 3695) by theaddition of 25 μl FXIa substrate solution (Hematological TechnologiesInc., Catalog Number SN-13a) diluted 1:100 in buffer B) into each wellof the 96-well plate. Next, 25 μl of all samples prepared in the 1^(st)96-well plate (tested samples, samples for standard curve preparation,positive control and blanks samples) were transferred into the parallelwells in the second plate and the second plate was transferredimmediately into an ELISA reader (SpectraMax). The following parameterswere used for the reading: Recording for 15 minutes every 30 seconds;Excitation: 350 nm; Emission: 470 nm; Cutoff: 455 nm; Temperature: 37°C.; The shaker was activated 15 seconds before the first read; andV_(max) rate [Relative Fluorescence Unit (FRU)/min] was measured from0-900 seconds.

The V_(max) of the kinetic reaction is a calculation of the reaction,using a linear curve fit. A creeping iteration is performed using theV_(max) points and the slope of the steepest line segment is reported asV_(max) rate as the RFU (Relative Fluorescence Unit).

FXIa concentration (ng/ml) in the tested sample was extrapolated by thesoftware from a standard curve generated using FXIa (described above)taking into consideration sample dilution (the blank was subtractedautomatically).

Protein content was measured by the Biuret method using the Totalprotein reagent (Sigma Diagnostic INC., Catalog Number 541-2) accordingto the manufacturer's instructions.

IgG subclasses distribution was measured using BIND A RID kit for humanIgG subclasses Combi kit (The Binding Site Ltd.; Catalog Number RK021)according to the manufacturer's instructions.

Anti-Diphtheria Antibodies titer was measured by Diphtheria kit(VITROTECH) according to the manufacturer's instructions.

Hepatitis B Surface Antigen (anti-HBsAg) Antibody was measured by a kitobtained from Abbott Laboratories; Catalog Number: LBXHBS.

Measurements of Kallikrein Levels (Chromogenic Method).

Kallikrein levels were measured by a chromogenic method. In the method,kallikrein cleaves a specific chromogenic substrate. In the cleavagereaction the kallikrein substrate (which is composed from a chromogenicreporter group para-nitroaniline (pNa) attached to a Kallikreinsubstrate oligopeptide sequence) is hydrolyzed between the oligopeptidesequence and the pNa group. Once cleaved from the peptide moiety, p-Naexhibits a high absorbance at 405 nm. This activity is directly relatedto the amount of kallikrein in the sample. The observed absorbance ismeasured by using an ELISA reader at 405 nm. More specifically, themeasurement is carried out as follows:

First stage (1^(st) 96-well plate preparation)—Tested samples weretested either diluted 1:5 (in the case the sample is tested aftersubjecting the immunoglobulin solution to the cation exchanger ordiluted 1:30 (in the case the sample is tested before subjecting theimmunoglobulin solution to the cation exchanger). The dilution wascarried out in buffer A [20 mM Hepes, 150 mM NaCl, 0.1% w/v BSA]. 50 μlfrom the above mentioned samples were added into a well of a 96well-plate (Costar; Catalog Number 3797) in quadruplicate.

A standard curve was prepared using kallikrein (Enzyme ResearchLaboratories; Catalog Number HPKa-1303) diluted in buffer A to thefollowing concentrations: 400, 200, 100, 50, and 12.5 ng/ml. 50 μl ofeach concentration was added into the wells in quadruplicate. Twopositive controls (immunoglobulin preparations which contained about 70and 15 ng/ml of kallikrein) and blank samples (Buffer A) were added toeach plate in quadruplicate (50 μl of each positive and blank samples).

Second stage (2^(nd) 96-well plate preparation)—For the kinetic reactiona second 96-well plate was prepared (Costar; Catalog Number 3695) by theaddition of 25 μl kallikrein substrate solution {Biophen SC31(02)(HYPHEN BioMed, Catalog Number 229031—reconstituted in 5 ml of purifiedwater and then diluted 1:7 in buffer A} into each well of the 96-wellplate. Next, 25 μl of all samples prepared in the 1^(st) 96-well plate(tested samples, samples for standard curve preparation, positivecontrol and blanks samples) were transferred into the parallel wells inthe second plate and the second plate was transferred immediately intoan ELISA reader (SpectraMax). The following parameters were used for thereading: Recording for 15 minutes every 34 seconds; Absorbance: 405 nm;Temperature: 37° C.; The shaker was activated 15 seconds before thefirst read; and V_(max) rate [OD/min] was measured from 0-900 seconds.

The V_(max) of the kinetic reaction is a calculation of the reaction,using a linear curve fit. A creeping iteration is performed using theV_(max) points and the slope of the steepest line segment is reported asV_(max) rate as the Absorbance Units/min.

Kallikrein concentration (ng/ml) in the tested sample was extrapolatedby the software from a standard curve generated using Kallikrein(described above) taking into consideration sample dilution (the blankwas subtracted automatically).

Example 1 The Effect of pH Level of an Immunoglobulin Solution onEfficacy of Cation Exchange Chromatography to Remove FXIa from theSolution

The following experiment was aimed to determine the effect of the pHlevel of the immunoglobulin solution on FXIa removal from the solutionby a cation-exchanger.

The isoelectric point of FXIa is about 9 (Bonno B N, Griffin J H. Humanblood coagulation factor XI: purification, properties, and mechanism ofactivation by activated factor XII. J Biol Chem 1977; 252:6432-6437).Since a cation exchanger is used, a pH level of lower than theisoelectric point (wherein FXIa has a net positive charge) can be used.In this experiment, the effect of a pH level range of 4-7 was evaluated.

For this purpose, an immunoglobulin solution was prepared according tosteps a-c described in the Material and Methods section. In the nextstep, the pH of the solution was adjusted either with 0.5 N NaCl or with0.1 N NaOH to the desired tested pH (a pH range of 4-7; the pH level ofthe solution was measured using an electronic pH monitoring device.) and100 ml of each of the resulting immunoglobulin solutions was subjectedto the cation-exchanger column.

Sulfopropyl-column (SP-column) was used as the cation-exchanger.Column's preparation: 4 ml of SP resin (TOSOHAAS; Catalog NumberToyopearl, SP-650M) were mounted inside a 1 cm diameter column (Bio-Rad)achieving a bed height of 6 cm. The chromatography was carried out atroom temperature (22±2° C.) at a flow rate of 2 ml/min. The temperatureof the loaded solution was 22±2° C.

To evaluate the removal of FXIa, the level of FXIa was measured in theloading material (“Load”) and after collecting the solution from thecolumn (“Un-bound”). The measurement was carried out by the TG assay asdescribed in the Materials and Methods section. In this experiment,thrombin generation values in the loading material were in the range of246-271 nM.

To estimate the IgG recovery in the un-bound fraction, the total proteinrecovery (%) was measured (the IgG consists of about 95% of the totalprotein). The results are presented in Table 1 below.

The isoelectric point of IgG is 6-8. Thus, at a pH level of lower than 6the IgG (which has a positive net charge) may be bind to the cationexchanger as well (in addition to the FXIa) thereby resulting in low IgGrecoveries.

TABLE 1 The effect of pH level of an immunoglobulin solution on theefficacy of a cation-exchanger to remove FXIa from the solution and onthe total protein recovery. pH level of the Thrombin Total proteinloading material Sample (nM) recovery (%)* 4 Un-bound 40.5 97.2 4.6Un-bound 42 92.6 5.3 Un-bound 48.7 91.1 5.7 Un-bound 68.9 92.1 6.1Un-bound 71 92.8 6.5 Un-bound 102.6 98.1 7 Un-bound 110.8 94 *Theevaluation was carried out by comparing the protein content (using theBiuret method) before (“load”) and after (“un-bound”) subjecting thesolution to the column.

The results show that pH levels of higher than 6 resulted in poor FXIaremoval (high TG values) and pH levels of lower than about 6 resulted ina high removal of FXIa (low TG values) from the immunoglobulin solutionand at the same time with a high IgG recovery (as measured by the totalprotein recovery). These results are surprising since at pH level lowerthan 6, the IgG (having an isoelectric point of 6-8) has a positive netelectrical charge and as such was expected to bind to the negativelycharged groups of the cation-exchanger column and thus low IgG recoverywere expected.

Another set of experiments were carried out using a carboxymethyl(CM)-column (a weak cationic exchanger obtained from TOSOHAAS; CatalogNumber Toyopearl, CM-650M). The experiment was carried out in the sameconditions as the SP-column. With the CM-column only a low pH range of4-5.5 was examined. The results were comparable with the resultsobtained with the SP-column showing that at the tested pH (4-5.5) a highremoval of FXIa and at the same time a high IgG recovery was obtained.The resin's volume was 8 ml; the column's diameter was 1 cm and the bedheight was 10 cm.

Example 2 The Effect of pH Level of an Immunoglobulin Solution onEfficacy of Mustang® S Membrane (a Cation-Exchanger) to Remove FXIa fromthe Solution

The following experiment was aimed to examine the effect of low pHlevels of the IVIG solution on FXIa removal from the solution by usingMustang® S membrane cation-exchanger [coin filters; Membrane Bed Volume(MV)=0.35 ml; Pall; Catalog Number NP8MSTGSP1)]. In this experiment theeffect of a pH level range of 3.8-5.3 of the immunoglobulin solution onFXIa removal was evaluated.

For this purpose, an immunoglobulin solution was prepared according tosteps (a) through (e) described above in the Materials and Methodssection. In the next step, the pH of the diafiltrated solution wasadjusted with 0.5 N NaCl or 0.1 N NaOH to the desired tested pH (a pHrange of 3.8-5.3) and 30 ml of each of the resulting immunoglobulinsolutions was subjected to the Mustang® S membrane. The chromatographywas carried out at room temperature (22±2° C.) at a flow rate of 1.5ml/min. The temperature of the loaded solution was 22±2° C.

The level of FXIa was measured in the immunoglobulin solution before(“Load”) and after (“Un bound”) subjecting the solutions to thecation-exchanger. The evaluation was carried out by a fluorogenic methodas described in the Materials and Methods section under “Measurements ofFXIa levels”. The results are presented in Table 2 below.

TABLE 2 The effect of pH level of an immunoglobulin solution on theefficacy of a cation-exchanger to remove FXIa. pH level of the FXIa(ng/ml) loading Using a fluorogenic method FXIa removal material LoadUn-bound (%)* 3.8 270.5 129.2 52.24 4 302.2 34.5 88.58 4.3 422.4 50.188.09 4.5 360.2 67.8 81.18 4.7 312.7 53.9 82.76 5 367.8 76.6 79.17 5.3340.9 81.4 76.12 *Calculated by comparing the FXIa levels before(“load”) and after (“un-bound”) subjecting the solution to the column.

The results obtained show that pH levels higher than 3.8 resulted inefficient removal of FXIa from the immunoglobulin solution with theoptimal pH level being at a range of 4.0 to 5.0 (pH in which the highestpercentage of FXIa removal was observed).

In another set of experiments the effect of a higher pH level wasevaluated (up to 7.4). The experiment was carried out in the same manneras the previous experiment except that 20 ml immunoglobulin solutionswas loaded to the cation-exchanger (Mustang® S membrane); and thefiltration was carried out at a higher flow rate of 2.5 ml/min.

The level of FXIa was measured in the loading material (“Load”) and inun-bound fraction 10 (10 fractions of 2 ml were collected and themeasurement was carried out on fraction 10). The measurement was carriedout as above by the fluorogenic method. The results are presented inTable 3 below.

TABLE 3 The effect of pH level of an immunoglobulin solution on theefficacy of Mustang ® S membrane to remove FXIa. pH level of FXIa(ng/ml) the loading Using a fluorogenic method FXIa removal materialLoad Un-bound fraction 10 (%)* 5 116.7 28.3 75.75 6 115.6 68.5 40.74 7.4112.1 144.5 — *Calculated by comparing the FXIa levels before (“load”)and after (“un-bound”) subjecting the solution to the column.

Surprisingly, the results obtained show that although FXIa has apositive net charge in a pH level of lower than about 9 (its isoelectricpoint is 8.9-9.1), inefficient FXIa removal was obtained at pH of 6 andhigher.

Example 3 The Effect of Fluid Flow Rate on the Removal of FXIa from anImmunoglobulin Solution by Cation-Exchange Chromatography

The following experiment examines the effect of fluid flow rate on theremoval of FXIa from an immunoglobulin solution by a cation-exchangechromatography. The following flow rates were evaluated: 0.7, 1, 1.5,2.5 ml/min. The starting immunoglobulin solution used was as in Example2 and the same Mustang® S membrane was used as the cation-exchanger.

In view of the previous Examples, the pH of the immunoglobulin solutionwas adjusted to 4.2 (for an immunoglobulin solution loaded at a flowrate of 0.7, 1 and 2.5 ml/min) or 4.3 (for an immunoglobulin solutionloaded at a flow rate of 1.5 ml/min) prior to loading the immunoglobulinsolution to the cation-exchanger. The chromatography was carried out atroom temperature (22±2° C.), the temperature of the solution loaded onthe cation exchanger was 22±2° C. and a volume of 30 ml immunoglobulinsolution was applied to the cation-exchanger.

The level of FXIa was measured in the immunoglobulin solution before(“Load”) and after (“Un bound”) loading the solutions to thecation-exchanger. The measurement was carried out directly by thefluorogenic method and indirectly by the TG assay as described bove inthe Material and Method section. In this experiment, thrombin generationvalues in the loading material were in the range of 179-290 nM.

The results are presented in Table 4 below.

TABLE 4 The effect of fluid flow rate during cation-exchangechromatography on the removal of FXIa from an immunoglobulin solution.Thrombin (nM) Flow rate of the FXIa (ng/ml) Using FXIa Using the TGloading material a fluorogenic method removal assay (ml/min) LoadUn-bound* (%)*** Un-bound* 0.7 342.6 40.8 88.09 43.0 1.0 283.5 45.184.09 25.2 1.5 422.4 50.1 88.14 0.0 2.5 304.0 80.7 73.45 63.3 *For aflow rate of 0.7, 1, and 2.5 ml/min—15 fractions were collected (eachcontained 2 ml) and for the evaluation of FXIa levels a mixture ofunbound fractions: 2, 5, 7, 10 and 13 was used. For a flow rate of 1.5ml/min—the entire un-bound fraction was used for the evaluation. **Sincethe Mustang's Membrane Bed Volume (MV) is 0.35 ml-0.7 ml/min = 2 MV/min;1 ml/min = 2.9 MV/min; 1.5 ml/min = 4.3 MV/min; and 2.5 ml/min = 7.1MV/min. ***Calculated by comparing the FXIa levels before (“load”) andafter (“un-bound”) subjecting the solution to the column.

It was observed, according to the fluorogenic method, that the optimalflow rate for removal FXIa from an immunoglobulin solution was in therange of 0.7-1.5 ml/min (the highest FXIa removal percentage wasobserved in this flow rate range). It is also observed that according tothe TG assay, the optimal flow rate for FXIa removal from animmunoglobulin solution (the lowest thrombin level obtained in theun-bound fraction as compared to the other tested flow rates) was 1.5ml/min.

These results indicate that in order to further improve FXIa removalfrom an immunoglobulin solution a flow rate of lower than 2.5 ml/minsuch as a flow rate in the range of 0.7 to 1.5 ml/min (2-4.3 MV/min) canbe used during subjection of the solution to the cation-exchangermembrane.

Example 4 The Effect of Temperature Level of an Immunoglobulin Solutionon Efficacy of FXIa Removal by Cation-Exchange Chromatography

The following experiment examines the effect of temperature level of animmunoglobulin solution on FXIa removal by cation-exchangechromatography. For this purpose the immunoglobulin solution wasequilibrated to the following temperatures: 7° C., room temperature(22±2), and 37° C. The starting immunoglobulin solution used was as inExample 2 and the same Mustang® S membrane was used as thecation-exchanger.

According to the results obtained in Examples 2 and 3, the pH of theimmunoglobulin solution was adjusted to 4.2 prior to subjecting thesolution to the cation-exchanger, and the immunoglobulin solution wasloaded to the cation-exchanger at a flow rate of 1-1.5 ml/min. Thechromatography itself was carried out at room temperature (22±2° C.),and a volume of 30 ml immunoglobulin solution equilibrated to thedifferent temperatures was applied to the cation-exchanger.

The level of FXIa was measured in the immunoglobulin solution before(“Load”) and after (“Un-bound”) subjecting the solutions to thecation-exchanger. As in Example 3, the evaluation was carried out by thefluorogenic method and the TG assay as described in the Material andMethod section. The results are presented in Table 5 below. In thisexperiment, thrombin generation values in the loading material were inthe range of 251-292 nM.

TABLE 5 The effect of temperature level of an immunoglobulin solution onthe efficacy of a cation-exchanger to remove FXIa. Temperature level ofthe FXIa (ng/ml) loading Using a Thrombin (nM) material fluorogenicmethod FXIa removal Using the TG assay (° C.) Load Un-bound (%)*Un-bound  7 283.9 39.0 86.26 24.9 22 ± 2 283.5 45.1 84.09 25.2 37 274.249.2 82.05 59.7 *Calculated by comparing the FXIa levels before (“load”)and after (“un-bound”) subjecting the solution to the column.

The results obtained show that all tested temperature levels resulted inremoval of FXIa from the immunoglobulin solution (as compared to theloading material) with the optimal temperature being from roomtemperature (22±2° C.) down to 7° C.

Example 5 The Effect of Carrying Out a Second Cation-ExchangeChromatography Step on the Removal of FXIa from an ImmunoglobulinSolution

The following experiment examines whether carrying out a secondcation-exchange chromatography step would further increase FXIa removalfrom an immunoglobulin solution.

The starting immunoglobulin solution used was as in Example 2. In thefollowing experiment a volume of 2600 ml of the solution was subjectedto Mustang® S capsule having an MV of 10 ml (Pall; Catalog NumberCLM05MSTGSP1); the pH of the loaded solution was adjusted to 4.2; andthe filtration was carried out at room temperature (22±2° C.) at a flowrate of 30 ml/min (=3 MV/min). The temperature of the loaded solutionwas 22±2° C.

Six filtrate fractions (400 ml per fraction) were collected and FXIalevel was measured using the TG assay as described in the Material andMethod section. In the next step, all six filtrate fractions werecollected, pooled and subjected to a second filtration step through anew Mustang® S capsule (MV=10 ml). Again, six filtrate fractions (400 mlper fraction) were collected and FXIa level was measured using the TGassay. The results of the loading material and each filtrate fraction(1-6) in each filtration step are summarized in Table 6 below. Also, inorder to estimate the IgG recovery, the total protein recovery wasmeasured following the second cation-exchange chromatography step.

TABLE 6 The effect of carrying out a second cation-exchangechromatography step on the removal of FXIa from an immunoglobulinsolution. Thrombin (nM) Sample Using the TG assay First CapsuleFiltration Loading material 248.6 Un-bound (UB) fraction 1 0.0 UBfraction 2 15.2 UB fraction 3 40.0 UB fraction 4 62.5 UB fraction 5 84.8UB fraction 6 121.5 Second Capsule Filtration Loading material 74.9 UBfraction 1 0.3 UB fraction 2 0.0 UB fraction 3 0.0 UB fraction 4 2.1 UBfraction 5 0.0 UB fraction 6 0.0

It was observed that carrying out a second filtration step resulted inan increased removal of FXIa from the immunoglobulin solution ascompared to carrying out a single filtration step. It is also shown thesecond filtration did not alter the high IgG recovery (a total proteinrecovery of 91.6% was obtained following the second filtration step).

These results indicate that in order to obtain maximal removal of FXIafrom the immunoglobulin solution while substantially preserving the IgGlevels, the solution can be subjected to a cation-exchanger more thanonce.

Example 6 The Efficacy of an Affinity Column Chromatography in RemovingFXIa from an Immunoglobulin Solution

In Examples 1-5 it was shown that a cation-exchange chromatography wasan effective step for removing FXIa from an immunoglobulin solution at apH range of 3.8 to lower than 6. In the following example, the efficacyof an affinity column chromatography in the removal of FXIa wasexamined. Benzamidine Sepharose-column (Benzamidine Sepharose bindsserine proteases such as FXIa) was used as the affinity column.

For this purpose, an immunoglobulin solution was prepared according tostep a-c as described in the Material and Methods section. In the nextstep, the solution was subjected to the Affinity column. The followingconditions were employed in the affinity chromatography: 120 ml solutionwas loaded to the column; the process was carried out at roomtemperature (22±2° C.); the pH of the immunoglobulin solution was 7.4; afluid flow rate of 1.3 ml/min was used; the temperature of the subjectedsolution was 22±2° C. The column was prepared according to themanufacturer's instructions-5 ml of benzamidine sepharose (GE healthcareCatalog Number 17-5123-01) were mounted inside a 1 cm diameter column(Bio-Rad). Prior to use the column was washed with 5 CV of purifiedwater and equilibrated with 5 CV Buffer (50 mM Citrate and 0.2 M NaCl)having a pH of 7.4.

Three filtrate samples (40 ml per fraction) were collected and FXIalevel was measured in the loading material and in the filtrate sampleusing the fluorogenic method as described above in the Material andMethod section. The results are presented in Table 7 below.

TABLE 7 The effect of para-amino benzamidine affinity columnchromatography in the removal of FXIa from an immunoglobulin solution.FXIa (ng/ml) Sample Using a fluorogenic method Loading material 363.9Un-bound (UB) fraction 1 90.6 UB fraction 2 267.1 UB fraction 3 290.5

It was observed that in the first un-bound (UB) fraction the column wascapable of effectively binding FXIa. However, in the second and third UBfractions, FXIa was detected at high quantities in the immunoglobulinsolution.

These results indicate that under the used condition BenzamidineSepharose affinity chromatography is not suitable for effectivelyremoving FXIa from an immunoglobulin solution.

In the above experiment it was found that in the tested conditionsBenzamidine Sepharose affinity column was not effective in removingFXIa. In the following set of experiments heparin-affinitychromatography was tested (heparin was tested due to its ability to bindFXI and FXIa). In this experiment, the effect of different pH levels ofthe immunoglobulin solution was evaluated (in the range of 5.3-8).

For this purpose, an immunoglobulin solution was prepared according tosteps (a) through (c) described above in the Material and Methodsection. In the next step, the pH of the solution was adjusted eitherwith 0.5 N NaCl or with 0.1 N NaOH to the desired tested pH and 150 mlof each of the immunoglobulin solutions with different pH were subjectedto the heparin-affinity chromatography.

Column's preparation: 8 ml of Capto Heparin resin (GE healthcare) weremounted inside a 1 cm diameter column (Bio-Rad) achieving a bed heightof 10 cm. The chromatography was carried out at room temperature (22±2°C.) at a flow rate of 2 ml/min. The temperature of the loaded solutionwas 22±2° C. The column was equilibrated, prior to loading theimmunoglobulin solution, with 40 ml of 50 mM citrate buffer having a pHlevel corresponding to the pH level of the immunoglobulin solution to beloaded (i.e. a pH level of 5.3, 6.5, 7.4, and 8).

Four filtrate fractions (40 ml per fraction) were collected and FXIalevel was evaluated in the loading material and in the filtratefractions using the fluorogenic method as described above. The resultsare presented in Table 8 below.

TABLE 8 The effect of heparin - affinity chromatography carried out atdifferent pH levels of an immunoglobulin solution on the efficacy toremove FXIa from the solution. FXIa (ng/ml) pH level of the Using afluorogenic loading material Sample method 5.3* Load 192.87 Un-bound(UB) fraction 1 25.51 UB fraction 2 28.92 UB fraction 3 40.85 UBfraction 4 201.48 6.5 Load 201.8 Un-bound (UB) fraction 1 38.7 UBfraction 2 47.9 UB fraction 3 55.2 UB fraction 4 190.4 7.4 Load 214.2 UBfraction 1 51.0 UB fraction 2 64.6 UB fraction 3 107.0 UB fraction 4159.6 8 Load 209.9 UB fraction 1 65.4 UB fraction 2 78.7 UB fraction 3112.7 UB fraction 4 167.0 *In pH 5.3 the following conditions were used(instead of the conditions specified for the rest of the pH levels): 8ml Heparin-Hyper DM (GE Healthcare Life Sciences) was used; and theloading volume was 125 ml. Equilibration prior to loading was carriedout with 40 ml buffer (containing 50 mM citrate and 0.2M NaCl at pH 7.4)at a fluid flow rate of 2 ml/min. In general, it was observed thatbinding of FXIa to the heparin resin improved by decreasing the pH ofthe immunoglobulin solution. The best results were observed on the firstthree unbound fractions of the lowest pH (5.3). However, after about 15column volume, FXIa is collected in the filtrate (relatively high FXIaquantity in UB fractions 4 of pH 5.3 was observed).

Example 7 The Effect of pH Level of an Immunoglobulin Solution onEfficacy of an Anion-Exchange Chromatography to Remove PKA from theSolution

The following experiment was aimed to determine the effect of the pHlevel of an immunoglobulin solution on PKA removal from the solution byan anion-exchanger.

In this experiment the effect of a pH level range of 6.4-8.2 wasevaluated. The pH level of the solution was measured using an electronicpH monitoring device.

DEAE-column (Toyopearl DEAE-650M; TOSOHAAS) was used as theanion-exchanger. 11 ml resin was used. The column's diameter was 1 cm,and a bed height of 14 cm was used.

In this experiment an immunoglobulin solution prepared from paste II andsubjected to CUNO filtration in the manner described in the Materialsand Methods section (an immunoglobulin solution following step b) wasused. In the next step, the pH level of the solution was adjusted with0.5 N HCl or 0.5 N NaOH to the desired tested pH level (a pH range of6.4-8.2) and 200 ml of each of the resulting immunoglobulin solutionswas subjected to the anion-exchange chromatography (step c of theimmunoglobulin solution preparation described in the Materials andMethods section) (in all experiments, 200 ml containing about 70 mg/mlprotein were subjected to the anion-exchange chromatography). Thechromatography step was carried out at 8° C.; the temperature of theloaded immunoglobulin solution was 8° C.; and a linear fluid flow rateof 1.65 ml/min/cm² was used.

In Example 7 through 11, the same immunoglobulin solution was used asthe loading material (“Load”), i.e. the PKA level of the loadingmaterial was identical in all experiments.

The level of PKA and other characteristics of the immunoglobulinsolution such as the total protein recovery, IgG subclassesdistribution, the titer levels of anti-HBsAg and Anti-DiphtheriaAntibodies were evaluated in the “un-bound” fraction (=after subjectingthe solutions to the anion exchanger). The evaluations were carried outas described in the Materials and Methods section. The results aresummarized in Table 9.

TABLE 9 The effect of pH level of an immunoglobulin solution on theefficacy of DEAE-column to remove PKA. Anti- pH level of Total proteinIgG subclasses distribution Diphtheria the loading Recovery** Anti-HBsAgPKA (%) Antibodies material (%) (mIU/ml) (IU/ml) IgG1 IgG2 IgG3 IgG4(IU/ml) 6.4 97 4762 14.4 ND ND ND ND ND 7.0 97 4884 <LOQ 62.9 29.5 6.70.9 6.1 7.6 95 3450 <LOQ 63.6 28.8 6.6 1.0 5.8 8.2 97 2481 <LOQ 64.728.7 5.7 0.9 5.3 * ND—Not Determined; LOQ—limit of quantitation - avalue of <6. **Total protein recovery (%) was calculated by comparingthe protein content (using the Biuret method) before and aftersubjecting the solution to the column.

It was observed that a low pH level of 6.4 resulted in an increased PKAcontent in the recovered IgG solution as compared to the higher pHlevels. It was also observed that a pH level in the range of 7-8.2 (arange wherein the PKA has a zero net electrical charge and as such isnot expected to bind to positively charged groups) resulted in anincreased capacity of the anion-exchanger to remove PKA (the PKA levelwas below the limit of quantitation). The results also show that a pHlevel range of 7-8.2 also resulted in high protein recoveries in theun-bound fraction (a pH level wherein the IgG has a net zero or negativecharge and it was expected that some of it would be bound to the anionexchanger) (protein recoveries in the range of 95-100% were obtained),in unaltered IgG subclass distribution characteristics [typical IgGsubclasses distribution (IgG1—about 65%, IgG2—about 30%, IgG3—about 6%,and IgG4—about 1%), and typical anti-HBsAg (in the range of 2000-5000mIU/ml) and anti-Diphtheria antibody titres (about 6 IU/ml)].

These results indicate that the immunoglobulin solution should have a pHlevel range of 7 to 8.2 while subjected to the anion-exchanger in orderto efficiently remove PKA from the solution.

Example 8 The Effect of Temperature Level on PKA Removal from anImmunoglobulin Solution by Anion-Exchange Chromatography

The following experiment examines the effect of temperature level of animmunoglobulin solution on PKA removal from the solution by ananion-exchanger Immunoglobulin solutions equilibrated to the followingtemperatures were evaluated: 2, 8, 14 and 20° C. The startingimmunoglobulin solution (loading material) used and a DEAE-column wereused as in Example 7. The chromatography step itself was carried out at8° C.; a linear fluid flow rate of 1.65 ml/min/cm² and a pH level of 7.5were used; and the loading volume was 200 ml.

The level of PKA and other characteristics of the immunoglobulinsolution (same parameters as above) were evaluated in the un-boundfraction. The results are summarized in Table 10.

TABLE 10 The effect of the temperature of an immunoglobulin solution onthe efficacy of DEAE-column to remove PKA. Temperature level of theTotal Anti- loading protein IgG subclasses distribution Diphtheriamaterial recovery PKA Anti-HBsAg (%) Antibodies (° C.) (%) (IU/ml)(mIU/ml) IgG1 IgG2 IgG3 IgG4 (IU/ml) 2 100 7.7 4616 ND ND ND ND ND 8 96<LOQ 3222 65.4 27.0 6.5 1.1 6.4 14 97 <LOQ 3620 65.0 27.8 6.3 1.0 6.4 20100 <LOQ 4970 64.1 27.8 7.0 1.1 6.4 * ND—not determined; LOQ—limit ofquantitation - a value of <6.

It was found that an IgG solution loaded having a low temperature of 2°C. resulted in an increased PKA content in the recovered unboundfraction. A temperature in the range of 8 to 20° C. resulted in anoptimal PKA removal (the PKA level was below the limit of quantitation),with unaltered IgG content and IgG subclass distribution characteristicsfollowing the DEAE chromatography step, and typical anti-HBsAg andanti-Diphtheria antibody titres (see typical values above).

Example 9 The Effect of the Loading Volume of an Immunoglobulin Solutionon Efficacy of an Anion-Exchange Chromatography to Remove PKA

The following experiment examines the effect of the loading volume of animmunoglobulin solution on PKA removal from the solution by ananion-exchanger. The following volumes were loaded onto the column: 12column volume (CV), 20 CV, and 25 CV (the resin's volume and thecolumn's premasters are as in Example 7). By “12 column volume” it ismeant 12 times the resin's volume (which was 11 ml).

The starting immunoglobulin solution (loading material) used was as inExample 7 and a DEAE-column was used as the anion-exchanger. Thechromatography step was carried out at 8° C.; according to the precedingExamples, the temperature of the loaded immunoglobulin solution was 8°C. and the pH was 7.5; and a linear fluid flow rate of 1.65 ml/min/cm²was used.

The level of PKA and other characteristics of the immunoglobulinsolution (same parameters as above) were evaluated in the un-boundfraction. The results are summarized in Table 11.

TABLE 11 The effect of increasing loading volumes of an immunoglobulinsolution on the efficacy of a DEAE-column to remove PKA. Total Loadprotein IgG subclasses distribution Anti- volume recovery PKA Anti-HBsAg(%) Diphtheria (CV) (%) (IU/ml) (mIU/ml) IgG1 IgG2 IgG3 IgG4 (IU/ml) 12100 <LOQ 5107 63.2 29.2 6.6 1.0 6.5 20 93 <LOQ 3727 64.8 27.9 6.4 0.96.4 25 99 <LOQ 5329 66.6 26.0 6.4 1.1 6.1 * LOQ—limit of quantitation -a value of <6.

The results show that in all tested loading volumes the DEAE-columnefficiently removed PKA substantially without impairing proteinrecoveries (a total protein recovery in the range of 93-100% wereobtained), IgG characteristics (all the values are comparable to thetypical values or anti-Diphtheria and Anti-HBsAg titers (see the typicalvalues in the Example 7).

Example 10 The Effect of the Linear Velocity of the ImmunoglobulinSolution During Subjection to an Anion-Exchange Chromatography onEfficacy of the Chromatography to Remove PKA

The following example examines the effect of linear velocity of thesolution during loading on the efficacy of the anion-exchangechromatography to remove PKA. The following linear velocities wereevaluated: 1, 2, 3, and 4 cm/min/ml.

The loading material used and the DEAE-column used were as in Example 7.The chromatography step was carried out at 8° C.; the temperature of theloaded immunoglobulin solution was 8° C.; a pH of 7.5 was used; and theloading volume was 200 ml.

The level of PKA and other characteristics of the immunoglobulinsolution (same as above) were evaluated in the un-bound material. Theresults are summarized in Table 12.

TABLE 12 The effect of linear velocity of an immunoglobulin solution onthe efficacy of a DEAE-column to remove PKA. Linear velocity of TotalAnti- the loading Protein IgG subclasses distribution Diphtheriamaterial Recovery Anti-HBsAg PKA (%) Antibodies (cm/min/ml) (%) (mIU/ml)(IU/ml) IgG1 IgG2 IgG3 IgG4 (IU/ml) 1 97 3870 <LOQ 65.5 27.4 6.1 1.0 5.32 96 3844 <LOQ 65.5 27.0 6.4 1.1 5.2 3 98 3739 11.2 ND ND ND ND ND 4 1013751  8.6 ND ND ND ND ND * ND—not determined; LOQ—limit ofquantitation - a value of <6.

The results show that a linear velocity between 1 and 2 cm/min/mlefficiently removed PKA from the immunoglobulin solution withoutimpairing protein recovery or IgG characteristics. Higher linearvelocities (e.g. 3-4 cm/min/ml) resulted in lower PKA removal.

Example 11 Removal of Thrombogenic Agents from an ImmunoglobulinSolution by Using Tandem Ion-Exchange Chromatography

An immunoglobulin solution was prepared from paste II and subjected toCUNO filtration in the manner described in the Materials and Methodssection (an immunoglobulin solution following step b). In the next step,the solution was subjected to a DEAE-column [(Toyopearl DEAE-650M;TOSOHAAS) (11 ml resin was used; the column's diameter was 1 cm, and abed height of 14 cm was used)] under the following conditions: thechromatography step was carried out at 8° C.; the temperature of theloaded immunoglobulin solution was 8° C.; a linear fluid flow rate of1.65 ml/min/cm²; and the pH level of the loaded immunoglobulin solutionwas equilibrated to 7.5. The volume of the loaded solution was 200 ml.The column was equilibrated prior to loading the solution as describedin the Materials and Methods section.

The level of PKA and other characteristics of the immunoglobulinsolution (as above) were evaluated in the“un-bound” fraction (=aftersubjecting the solutions to the anion exchanger). The measurements werecarried out as described in the Materials and Methods section. Theresults are summarized in Table 13.

TABLE 13 PKA removal from an immunoglobulin solution by ananion-exchanger. Total Protein Re- Anti- IgG subclasses Anti- PKAcoveries HBsAg (%) Diphtheria (IU/ml) (%) (mIU/ml) IgG1 IgG2 IgG3 IgG4(IU/ml)* <LOQ 100 4332 63.6 28.9 6.6 0.9 6.7 LOQ—limit of quantitation -a value of <6.

The results show that under the used conditions, the DEAE-columnefficiently removed PKA substantially without impairing protein recovery(a total protein recovery of 100 was obtained), IgG characteristics (allthe values are comparable to the typical values), anti-Diphtheria andanti-HBsAg titers (see the typical values in the Example 7).

Following contacting the immunoglobulin solution with theanion-exchanger, the collected un-bound fraction is subjected to stepsd)-e) of the immunoglobulin preparation as described in the Material andMethods section. In the next step, the solution is subjected to a cationexchanger under the following conditions: a pH level in the range of 3.8to 6; a flow rate of lower than 2.5 ml/min [e.g. a flow rate in therange of 0.7 to 1.5 ml/min (2-4.3 MV/min)]; and the temperature of theloaded immunoglobulin composition is in the range of from roomtemperature (22±2° C.) down to 7° C.

FXIa removal is evaluated in the loading material (“Load”) and aftercollecting the solution from the column (“Un-bound”). The measurement iscarried out by the TG assay and/or by the fluorogenic method asdescribed in the Materials and Methods section.

Example 12 The Effect of SDR-Column on FXIa Removal from anImmunoglobulin Solution

The following experiment was aimed to determine the ability ofSDR-column to remove FXIa from an immunoglobulin solution.

For this purpose, an immunoglobulin solution was prepared from paste IIaccording to step a)-e) in the manner described in the Materials andMethods section. In the next step, the solution was subject to acation-exchange chromatography using Mustang® S capsule as follows(scale-up conditions):

Prior to subjecting the immunoglobulin solution to the cation-exchanger,the solution was filtered through 0.2 μm CA filter obtained from Corning(in order to reduce aggregates). The Mustang® filtration was carried outthrough two Mustang® S filters which were connected in series. Prior tothe Mustang® filtration, the two filters were washed separately with 30Kg of 1 N NaOH at a flow rate of 2.1 L/min (for both filters). As asecond wash, both filters were washed separately with 50 Kg of 1 N NaClat a flow rate of 1.8 L/min (for the first filter) or at a flow rate of2.1 L/min (for the second filter). Finally, the filters were washedseparately with 50 Kg of 20 mM Sodium Acetate (at a pH of 4.2) at a flowrate of 1.8 L/min (for the first filter) or at a flow rate of 1.9 L/min(for the second filter). The above washes were carried out to obtain apH level of 4.2. All washes were carried out at a pressure of 0.

Mustang® filtration: the immunoglobulin solution was filtered throughthe two washed Mustang® S filters at a flow rate of 1.6 L/min. A totalof 160 Kg immunoglobulin solution (un-bound fraction) was collected. Thetemperature of the loaded solution was 7° C. while the filter was atroom temperature. The pH of the loaded immunoglobulin solution was4.2-4.3.

In the next step, the filtrate (un-bound fraction) was subjected toSolvent/Detergent (S/D) treatment and SDR-column as described in theMaterials and Methods section.

The level of FXIa was measured in the solution before filtering thesolution through the Mustang S membrane (i.e. an immunoglobulin solutionprepared according to step a-e as described in the Material and Methodssection), after filtering the solution through the Mustang S membrane,and after subjecting the solutions to S/D treatment+SDR-column.

The evaluation was carried out by a fluorogenic method as described inthe Materials and Methods section. The results are presented in Table 14below.

TABLE 14 Removal of FXIa from an immunoglobulin solution by SDR-column.FXIa (ng/ml) Tested sample Using a fluorogenic method Pre-Mustang Sfiltration 529.4 Post-Mustang S filtration  37.6 Post SD treatment +SDR-column  3.9 (<LOD) * LOD—limit of detection.

As shown in Table 14, the addition of S/D treatment and S/D removal stepby SDR-column results in removal of residual amounts of FXIa.

Example 13 Removal of Kallikrein from an Immunoglobulin Solution byUsing Mustang® S Capsule (a Cation-Exchanger)

The following experiment was aimed to determine the ability of Mustang®capsule to remove kallikrein from an immunoglobulin solution.

For this purpose, an immunoglobulin solution was prepared from paste IIaccording to steps (a)-(e) in the manner described in the Materials andMethods section. In the next step, the solution was subject to theMustang® S capsule as follows (scale-up conditions):

Prior to the Mustang® filtration, each filter was pre-washed separatelywith at least 30 Kg of 1 N NaOH at a flow rate of 1.6-2.3 L/min. As asecond wash, each filter was washed separately with at least 50 Kg of 1N NaCl at a flow rate of 1.6-2.3 L/min. Finally, each filter wasequilibrated separately with 50 Kg of 20 mM Sodium Acetate (at a pH of4.2) at a flow rate of 1.6-2.3 L/min. The above washes were carried outto obtain a pH level of 4.2. All washes were carried out at a pressureof ≦1 bar.

Prior to subjecting the immunoglobulin solution to the cation-exchanger,the solution was filtered through 0.2 μm Durapore filter (Milipore) (inorder to reduce aggregates). The resulting immunoglobulin solution wasfiltered through two Mustang® S capsules (which were pre-washed andequilibrated as specified above) that were connected in series.

Mustang® filtration: the immunoglobulin solution was filtered throughthe two capsules at a flow rate of 1.6-2.3 L/min [about 160 Kg (about160 L) immunoglobulin solution was loaded; and about 70 mg/ml protein].A total of about 160 Kg immunoglobulin solution (about 160 L) (un-boundfraction) was collected.

The temperature of the loaded solution was about 7° C. while the filterwas at room temperature; filtration was carried out at RT; the pH of theloaded immunoglobulin solution was 4.1-4.3.

The level of Kallikrein removal was measured in the immunoglobulinsolution before (“Pre-filtration”) and after filtration(“Post-filtration”) through the Mustang® S capsule, and the percentageof Kallikrein removal was calculated. The evaluation was carried out bya chromogenic method as described in the Materials and Methods section.

To estimate the IgG recovery after filtration, the total proteinrecovery (%) was measured The obtained Kallikrein removal is shown inTable 15 below, and the obtained total protein recovery (%) is shown inTable 16.

TABLE 15 Kallikrein removal by Mustang ® S filters. Kallikrein RemovalSample V_(max)* (ng/ml) (%) Pre-filtration 478.2 3760.6 Post-filtration20.6 126.1 97%

TABLE 16 Protein recovery following Mustang ® S filtration. TotalProtein protein Recovery Sample (mg/ml) (%) Pre-filtration 65.4Post-filtration 63.4 97%

It was observed that under the specified conditions, loading animmunoglobulin solution to a cation exchanger resulted in 97% kallikreinremoval while 97% of the protein (IgG) was recovered.

Example 14 Checking the Thrombosis-Inducing Activity of anImmunoglobulin Solution Prepared According to the Invention in anIn-Vivo Model

The following experiment was aimed to examine whether an immunoglobulinsolution which was subjected to kallikrein, PKA and/or FXIa removal asin the preceding Examples exhibit reduced thrombosis-inducing activity.The evaluation was carried out using an in-vivo model as described inWessler et al. (Biologic assay of a thrombosis-inducing activity inhuman serum. J Appl Physiol. 1959; 14:943-946).

It was observed, using the Wessler animal model, that an immunoglobulinsolution subjected to PKA and/or FXIa removal according to the inventionexhibited reduced thrombosis-inducing activity.

What is claimed is:
 1. A method for preparing an immunoglobulincomposition comprising the steps of: a) contacting an immunoglobulinsolution obtained by Cohn Fractionation and/or Kistler-Nitschmannfractionation or a fraction thereof with an anion exchanger equilibratedat a pH in the range of 7 to 8.2, and a cation exchanger equilibrated ata pH in the range of higher than 3.8 to equal to or lower than 5.3 toallow prekallikrein activator (PKA) (“PKA”) to bind to the anionexchanger, and Factor XIa, Factor XI and/or Kallikrein to bind to thecation exchanger; and b) collecting the unbound fractions comprisingimmunoglobulin.
 2. The method according to claim 1, wherein the cationexchanger chromatography is carried out twice.
 3. The method accordingto claim 1, wherein the cation exchanger chromatography is carried outat a pH in the range of higher than 3.8 to equal to or lower than 5.0.4. The method according to claim 1, wherein the cation exchangerchromatography is carried out at a pH in the range of equal to or higherthan 4.0 to equal to or lower than 5.0.
 5. The method according to claim1, wherein the cation exchanger chromatography is carried out at a pH inthe range of higher than 3.8 to equal to or lower than 4.7.
 6. Themethod according to claim 1, wherein the cation exchanger chromatographyis carried out at a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, or4.7.
 7. The method according to claim 1, wherein the cation exchangerchromatography is carried out at a pH in the range of higher than 3.8 toequal to or lower than 4.3.
 8. The method according to claim 1, whereinthe cation exchanger chromatography is carried out at a pH in the rangeof equal to or higher than 4.0 to equal to or lower than 4.3.
 9. Themethod according to claim 1, wherein the cation exchanger chromatographyis carried out at a pH in the range of equal to or higher than 4.1 toequal to or lower than 4.3.
 10. The method according to claim 1, furthercomprising a negative chromatography using a chromatographic materialcomprising three-dimensional cross-linked hydrophobic acrylic polymer.11. The method according to claim 1, wherein the cation exchanger is inthe form of a membrane.
 12. The method according to claim 1, wherein thecation exchanger comprises a sulfonic functional group.
 13. Animmunoglobulin composition derived from blood or blood fractions,comprising a total protein level of 4%-10% and that is obtainedaccording to the method of claim
 1. 14. A receptacle containing theimmunoglobulin composition according to claim 13.